Evolution of resistance to HIV-1 reverse transcriptase inhibitors

Transcription

1 Evolution of resistance to HIV-1 reverse transcriptase inhibitors Marleen Huigen

2 ISBN: Cover: HIV-1 replicates as a viral quasispecies, which can be seen as a cloud of distinct but closely related genetic variants. Layout: K. van de Westelaken en M. Huigen Printed by: Gildeprint drukkerijen The printing of this thesis was financially supported by the J.E. Jurriaanse Stichting, the Utrecht University, Merck Sharp & Dohme B.V., Boehringer Ingelheim bv, Tibotec, a division of Janssen-Cilag and the Eijkman Graduate School for Immunology and Infectious Diseases.

3 Evolution of resistance to HIV-1 reverse transcriptase inhibitors Evolutie van resistentie tegen HIV-1 reverse transcriptase remmers (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. W.H. Gispen, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 11 oktober 2007 des middags te 2.30 uur door Cornelia Dirkje Geertruida Huigen geboren op 25 november 1979 te Tiel

4 Promotor: Co-promotoren: Prof. dr. J. Verhoef Dr. C.A.B. Boucher Dr. M. Nijhuis The research presented in this thesis was in part funded by European Union grant QLK2-CT and was in part financially supported by unrestricted institutional grant from Bristol-Myers Squibb.

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6 Beoordelingscommissie: Prof. dr. I.M. Hoepelman (voorzitter) Prof. dr. B. Berkhout Prof. dr. F. Miedema Prof. dr. P.J.M. Rottier Prof. dr. J. van Strijp Paranimfen: Loek de Graaf Jan Huigen

7 Contents Chapter 1 General introduction 9 Chapter 2 Low genetic barrier caused by interplay between 33 mutations explains early virological failure of a triple reverse transcriptase inhibitor regimen Chapter 3 Evaluation of four approaches to measure replication 47 capacity of reverse transcriptase inhibitor resistant HIV-1 variants Chapter 4 Evolution of a novel 5-amino-acid insertion in the β3-β4 65 loop of HIV-1 reverse transcriptase Chapter 5 Identification of a novel resistance (E40F) and 83 compensatory (K43E) substitution in HIV-1 reverse transcriptase Chapter 6 HIV-1 transmission cluster with M41L singleton 99 mutation and decreased transmission of resistance in newly diagnosed Swedish homosexual men Chapter 7 Compensatory fixation explains long term persistence of 115 the M41L change in HIV-1 reverse transcriptase in the absence of therapy in a large transmission cluster Chapter 8 General discussion 131 Nederlandse samenvatting 149 Acknowledgements 153 Curriculum vitae 155 List of publications 157

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9 Chapter 1 General introduction Published in part in modified form in: Antiviral Research 75, (2007) and in Antimicrobial Drug Resistance Handbook (D.L. Mayers, ed.), Humana Press, in preparation

10 Chapter 1 Human immunodeficiency virus type 1 (HIV-1) The human immunodeficiency virus type 1 (HIV-1) is an enveloped retrovirus belonging to the genus of lentiviridae and is identified as the primary etiologic agent of the acquired immunodeficiency syndrome (AIDS) in ,2. HIV-1 primarily infects CD4 + T-lymphocytes and macrophages. An acute HIV-1 infection is characterized by a burst of viral replication and immune activation 3-5. During the chronic phase of infection continuous viral replication will occur resulting in a relatively stable HIV-1 RNA level, whereas the number of CD4 + T-lymphocytes gradually declines. In the absence of antiretroviral treatment, an HIV-1 infection will usually lead to a decline of CD4 + T-lymphocytes below a critical level. Cellmediated immunity will be lost and the infected individual becomes susceptible to opportunistic infections and malignancies 6. Estimations at the end of 2006 are that 39.5 million individuals are infected with HIV worldwide, 2.9 million people died last year of AIDS whereas 4.3 million new infections are estimated 7. HIV-1 quasispecies Similar to other RNA viruses, HIV-1 replicates as a complex and dynamic population of mutants referred to as viral quasispecies The quasispecies can be seen as a cloud of distinct but closely related genetic variants, in which wild type is defined as the most fit and most frequent individual sequence. The size and constitution (heterogeneity) of these HIV-1 populations are the result of multiple viral and host factors. Viral factors The error-prone viral enzyme reverse transcriptase (RT) lacks 3 to 5 exonucleolytic proofreading activity, as opposed to DNA polymerases. It is estimated that HIV-1 RT generates 3x10-5 errors per base pair per replication cycle 13. Furthermore, the HIV-1 genome consists of two single-stranded RNA copies. RT can jump from one to the other RNA template resulting in a high frequency of recombinant viral DNA sequences Recombination can also occur when an individual is infected with two different HIV-1 strains 17. Other factors are the high turnover rates of the virions in an HIV-infected individual (10 7 to 10 9 viral particles per day) and of actively infected cells (in vivo half life of 1-2 days) And finally, an important factor influencing the size of the HIV-1 quasispecies within an individual is the viral replication capacity; i.e. the ability of a virus to produce infectious progeny in a specific environment

11 General introduction Host factors The viral quasispecies will also be influenced by several host factors, such as the genetic background of the host, the immune system, cellular restriction factors and target cell availability 21. This influence on the quasispecies can be direct or indirect by affecting HIV-1 replication. Several genetic factors such as a deletion in the CCR5 co-receptor (CCR5 32) and specific histocompatibility complex HLA types, e.g. HLA-B27, are associated with reduced susceptibility to HIV-1 infections and progression towards AIDS Cytotoxic T-lymphocytes (CTLs) have shown to decrease HIV-1 RNA levels in acute HIV-1 infection and continuous CTL activity has been measured during the chronic phase of infection 4,5, Viral escape mutations can be selected because they confer CTL escape by the prevention of proteasomal cleavage, a decrease in transporter associated with antigen processing (TAP) transport efficiency, prevention of MHC-1 binding or lowering CTL recognition 26, Similar to CTL escape, specific HIV-variants have been shown to escape neutralizing antibodies as well 35,36. Also, cellular host factors have been shown to affect the HIV-1 quasispecies. For instance, host cells harbour apolipoprotein B mrna editing enzyme catalytic polypeptide-like 3G (APOBEC3G) that deaminates deoxycytidines (dcs) to deoxyuridines (dus) in minus-strand DNA during reverse transcription 37,38. This results in a high-level G-to-A hypermutation of the proviral plus-strand cdna and subsequent abrogation of the viral replication. The viral accessory protein vif (virion infectivity factor) binds to APOBEC3G and induces its ubiquitination and subsequent degradation by the proteasome and thereby suppresses its incorporation in the virion. In this way, vif can reduce the G-to-A hypermutation to a non-lethal level, but cannot prevent all substitutions 37,38. Another recently identified cellular restriction factor that presents a novel and important innate immune defence is tripartite motif protein 5α (TRIM5α). This protein recognizes motifs within the capsid proteins and interferes with the uncoating process, and is thereby able to prevent HIV-1 infection. This can be prevented by binding of cellular cyclophilin A to the capsid protein 39,40. Finally, target cell availability will also affect HIV-1 replication, since an increase in CD4 + T-lymphocytes has been shown to increase the HIV-1 RNA load, whereas immunosuppressive treatment leading to a decline in target cells showed the opposite Evolution of HIV-1 Initially, because of the high HIV-1 RNA levels it was assumed that the HIV-1 population is infinite and that evolution of HIV-1 follows a predictable deterministic 11

12 Chapter 1 model Subsequent research revealed that the effective population size (N e ), defined as the average number of HIV-1 variants that produce infectious progeny, is relatively small 16, This can be explained because the majority of virus particles that are produced harbour deleterious mutations resulting in noninfectious viruses 53. Also, limited target cell availability and inactivation of potentially infectious viruses before they can produce progeny will reduce the effective population size. The N e is estimated at 10 3 to 10 4 which indicates that the population size is finite and follows a stochastic evolutionary pathway 16, In this model chance plays an important role and the adaptability of the HIV-1 quasispecies to changes in its environment is limited. Strong immune and drug pressure or transmission to a new individual can induce repeated bottlenecks and result in a loss in quasispecies heterogeneity and viral fitness. This evolutionary principle described as Muller s ratchet has been described extensively for RNA viruses including HIV-1 49, In conclusion, the limited effective population size and strong population bottlenecking suggest that HIV-1 will follow stochastic evolutionary principles. HIV-1 fitness and replication capacity Within a viral quasispecies every virus variant has a so-called viral fitness, which is originally defined as the replicative adaptability of a virus to its environment. It is estimated that almost every randomly selected virus clone in the population will have a mutation compared to wild type and will have a lower fitness level 46. The fitness of an HIV-1 population in an infected individual is mainly determined by the heterogeneity of the quasispecies, which is indirectly determined by its size. Darwin s concept of survival of the fittest drives selection and evolution of the viral quasispecies. This implies that one or more clones of the viral quasispecies will be selected, whereas others will be eliminated from the population due to the continuous replication, selection and competition that takes place. If the environment changes, for example during antiretroviral treatment, the viral quasispecies can change in response to this 10,12. In 1932, Sewall Wright described the classical concept of fitness landscape in which changes in viral fitness could be seen as movements of viral genomes through a rugged landscape of peaks and valleys 8. The HIV-1 quasispecies will always seek for an optimal viral fitness state for a given environment. This local optimal peak in the fitness landscape does not necessarily represent a global optimum. A population may be fixed at a certain fitness level because passage through a valley of lower-fitness intermediates to a more fit virus is not possible. Viral fitness is a complex parameter, dependent on numerous viral and host factors, that can be estimated by in vivo kinetics of a virus over time However, 12

13 General introduction the term viral fitness is difficult and often misused. Originally defined as the replicative adaptability of a virus to its environment or the ability of a virus to replicate under the selective forces present in its environment, current researchers often use the term viral fitness as the replication capacity of a virus in a given environment. But, fitness and replication capacity are not identical terms. Viral fitness includes not only the intrinsic replication capacity but is dependent on several other host and viral factors as well. All these factors cannot be taken into account when measuring viral fitness in vitro. Therefore, when estimating the in vitro fitness of specific virus variants, actually the intrinsic viral replication capacity is being measured and we will use this term accordingly. Several methods have been developed to measure in vitro replication capacity 12. First of all, a single cycle infection assay, which measures the number of infected cells or pseudotyped virus particles after a single round of replication Secondly, a multiple cycle assay in which viral replication is measured by the production of virus over time The third assay investigates the relative replication capacity in a replication competition experiment, in which two different viruses compete with each other in a dual-infection assay 16, The first two assays are much less time-consuming and laborious than the competition experiments, but small reproducible differences can only be detected in replication competition experiments. Depending on the research question the use of a virus isolate would be recommended, e.g. when studying the relation of replication capacity on pathogenesis. To determine the effect of particular mutations in RT on replication capacity the most optimal method is to generate a recombinant virus containing just (the polymerase encoding part of) RT without the putative influence of amino acid changes in the remaining part of the genome. The majority of in vitro replication capacity studies make use of recombinant virus clones, in which the gene (fragment) of interest is cloned in a wild type background. However, one can never exclude that other genomic regions outside the target sequence may have an effect on viral replication capacity as well 71. Reverse transcriptase and reverse transcriptase inhibitors All steps in the HIV-1 replication cycle can be considered as target for antiretroviral treatment. However, currently six different processes/enzymes can be inhibited by the anti-hiv-drugs that are approved by the U.S.A. Food and Drug Administration (FDA) or in clinical development: entry, reverse transcription, integrase, protease, maturation and fusion of the virus membrane with the host cell This thesis focuses on the viral enzyme reverse transcriptase and inhibitors that are directed against the process of reverse transcription. 13

14 Chapter 1 The viral enzyme reverse transcriptase (RT) is unique and essential for retroviruses and transcribes the viral genomic RNA into a complementary DNA (cdna) copy. Reverse transcription is a complex process and dependent on two distinct enzymatic activities of RT; a DNA polymerase that can use either RNA or DNA as template and a nuclease (Ribonuclease H or RNase H) specific for the RNA strand of RNA:DNA duplexes HIV-1 RT is a stable heterodimer consisting of two subunits of respectively 66 (p66) and 51 kda (p51) The p51 subunit is generated by proteolytic cleavage of the p66 subunit by the viral protease and lacks the C-terminal RNase H domain. Although the overall folding of the two subunits is similar, the spatial arrangement of the two subunits is completely different. The p51 subdomain adopts a closed formation and only plays a structural role, whereas the p66 subunit is organized to form a cleft into which the primer-template binds and represents the polymerase active site. Crystallographic studies show that the p66 subunit resembles a right hand grasping the primer template complex 82,83. Based on this 3D structure the enzyme has been divided into five distinct domains (Figure 1). These are the fingers, palm, thumb, connection domain and the RNase H subdomain at the carboxy-terminus. The latter subdomain cleaves the template RNA strand and degrades the transcribed RNA. The palm domain harbours the polymerase active site, located in a cleft formed by the flanking fingers and thumb subdomain, which play a role in positioning the template. The active site of RT contains three aspartic acids at amino acids 110, 185 and 186, which are involved in metal ion ligation and interact with the phosphates of the DNA primer and the incorporated nucleotides. These aspartic acids are conserved and required for the proper function of reverse transcription. The connection subdomain, as the name already implies, connects the polymerase and the RNAse H domain 76,79,84,85. Unlike most polymerases, HIV-1 RT lacks a 3-5 exonuclease activity resulting in a high variability in the enzyme (and the other genes) in both naive and treated patients. Apparently, RT can accept many amino acid changes without losing its enzyme activity 86. This deficit in proofreading also means that HIV-1 RT is hardly able to identify and excise inappropriate nucleotides once they are incorporated in the growing DNA chain. As a consequence, RT will also incorporate analogues of dntps 46,87. This property was used for the development of inhibitors directed against RT. In 1987 the FDA approved the first anti-hiv drug, the reverse transcriptase inhibitor zidovudine (AZT), a thymidine analogue with an azido group at the 3 position of the ribose. Subsequently, in the following years many other nucleoside analogues, dideoxynucleotides, were developed and introduced in the clinic. Nucleoside 14

15 General introduction analogues or nucleoside reverse transcriptase inhibitors (NRTIs) are analogues of the normal dntp substrates of DNA polymerase with important modifications. Fingers Template strand DNA Palm Primer strand DNA Thumb Connection RNase H p51 Figure 1. HIV-1 reverse transcriptase is a heterodimer consisting of two subunits: p51 and p66. The p51 subunit plays a structural role, whereas the p66 subunit is organized to form a cleft into which the primer-template binds and represents the polymerase active site. The p66 subunit resembles a right hand grasping the primer-template complex and can be divided into five domains: fingers, palm, connection, thumb and RNase H. These nucleoside analogues are administered as precursor compounds (prodrugs), which have to be tri-phosphorylated by host cellular kinases to their active form. After binding to the polymerase active site of RT they compete with the natural dntps for recognition as substrate (binding) and incorporation into the nascent DNA chain. Because nucleoside analogues lack the 3 -hydroxyl group on the ribose moiety they block further DNA synthesis, once they are incorporated. Currently, eight NRTIs are approved by the FDA to inhibit HIV-1 reverse transcription and thereby viral replication. Two thymidine analogues: zidovudine (AZT or 3' -azido -3' -deoxythymidine) and stavudine (d4t or 2', 3' -didehydro -2', 3' dideoxythymidine), three cytosine analogues: zalcitabine (ddc or 2', 3' - dideoxycytidine) which is rarely used anymore, lamivudine (3TC or (-)-β-l -2', 3' - dideoxy -3' -thyacytidine) and emtricitabine (FTC), the guanosine analogue 15

16 Chapter 1 abacavir (ABC) and the adenosine analogue didanosine (ddi or 2', 3' dideoxyinosine). The other adenosine analogue tenofovir disoproxil fumarate (TDF) is monophosphorylated and is designated a nucleotide analogue. Since both nucleoside and nucleotide RTIs act by the same mechanisms, the abbreviation NRTIs is used for both classes of compounds The other class of RTIs are the non-nucleoside reverse transcriptase inhibitors (NNRTIs). In contrast to NRTIs, these drugs act as non-competitive inhibitors by binding in the vicinity but not at the active site of RT. They do not have to be metabolized by cellular enzymes but directly target an allosterically located hydrophobic pocket at HIV-1 RT, thereby causing a conformational change that locks the catalytic site in an inactive conformation. In contrast to NRTIs, the NNRTIs are highly specific for HIV-1 and do not inhibit HIV-2 or other retroviruses. Three different NNRTIs are currently approved by the FDA: nevirapine (NVP), efavirenz (EFV) and delavirdine (DLV), although the latter drug is rarely prescribed anymore 89,91. Resistance to HIV-1 reverse transcriptase inhibitors Nucleoside and nucleotide reverse transcriptase inhibitor resistance Unfortunately, soon after the introduction of AZT monotherapy it became evident that HIV could develop an up to 100-fold increase in IC 50 towards this drug in patients receiving six months of treatment 92. The rapid selection of virus variants harbouring a resistance-associated mutation can be explained by the fact that HIV- 1 replicates as a quasispecies. Under drug pressure specific virus variants harbouring a mutation conferring a decrease in susceptibility will have an advantage over the others in the viral quasispecies and will be selected. Amino acid changes in the RT gene that confer NRTI resistance are shown in Table 1. These mutations are all located in the palm and fingers subdomains of HIV-1 RT (Figure 2) 46,80,93,94. Some substitutions are able to confer (high-level) resistance on their own. The M184V/I mutation confers resistance to 3TC, FTC, ABC and ddc, the L74V against ddi, ABC, ddc ( and TDF) and the K65R change confers resistance to d4t, ddi, ddc, 3TC, FTC, ABC and TDF 94,95. On the other hand, high-level resistance against AZT requires two or more mutations from the group of M41L, D67N, K70R, L210W, T215Y/F and K219Q/E 92,96,97. These mutations can be selected by d4t as well 98 and since AZT and d4t are both thymidine analogues, these mutations are referred to as thymidine analogue mutations (TAMs). Some researchers have divided TAMs into primary (K70R and T215Y/F) and secondary mutations (M41L, D67N, L210W and K219E/Q). The latter four mutations confer minimal resistance on their own, but increase the resistance level of the primary mutations HIV-1 develops these TAMs by two 16

17 General introduction distinct pathways; the TAM-1 pathway consisting of M41L, L210W, T215Y and sometimes D67N or the TAM-2 pathway including D67N, K70R, T215F and K219Q/E Because TAMs have been shown to confer cross-resistance against other nucleoside analogues as well, these changes are also referred to as nucleoside analogue mutations (NAMs). The accumulation of TAMs is considered one of the three multi-nrti resistance pathways. Figure 2. Location of NRTI and NNRTI resistance mutations. Q151M is a key amino acid change in another multi-nrti resistance pathway and is usually accompanied by the A62V, V75I, F77L and F116Y changes 106,107. Similar to the TAM-pathways the 151-complex results in a gradual increase in resistance caused by the accumulation of these changes. The third pathway to NRTI multi-drug resistance involves the appearance of amino acid insertions in the β3-β4 loop of RT (between amino acid 69 and 70). They are mainly polar dipeptides and are usually selected in the background of particular TAMs, especially M41L and T215Y/F. This insertion-complex confers high level resistance to AZT and intermediate to high level resistance to all approved NRTIs

18 Chapter 1 The mutations that are selected during NRTI treatment can be subdivided into two classes based on their mode of action. These mechanisms of nucleoside drug resistance are respectively called discrimination and excision. Discrimination results from the ability of RT to exclude the NRTI, but still be able to recognize the analogous dntp. This mechanism of resistance can be the result of respectively impaired binding of the nucleoside analogue compared to the dntp, an impaired incorporation of the nucleoside analogue or a combination of both 81,113. On genetic level discrimination is caused by amino acid substitutions as M184V/I, L74V, V75T, K65R and Q151M (Table 1). All these changes are located in or close to the substrate (dntp) binding site, which make it plausible that these mutations can affect the initial binding and/or positioning of the NRTI 80. The other mechanism of NRTI-resistance, excision, is the result of TAMs that cause an increase in a pyrophosphorolysis-like mechanism Pyrophosphorolysis is the reverse of the polymerization reaction. The RTprimer/template complex binds pyrophosphate (PPi), which attacks the monophosphate group linking the two last nucleotides of the primer strand and regenerates a dntp and a primer shortened by one nucleotide. In the situation that a nucleoside analogue is incorporated in the DNA chain and prevents further DNA synthesis, pyrophosphorolysis means that the 3 OH group on the primer will be free again when the nucleoside analogue is excised. Forward direction DNA synthesis can then resume 117. AZT resistant RT was able to unblock the AZT-terminated primer using ATP as PPi donor in contrast to wild type RT 118,122. This reaction, which is analogous to pyrophosphorolysis is named ATP- pyrophosphorolysis and is conferred by the TAMs. It is still not completely clear what the in vivo phosphate donor is although several reports have found that the nucleoside excision reaction is ATP-dependent 87, ATP-dependent primer unblocking can be inhibited by the dntp complementary to the next position on the template. When the primer is blocked with an NRTI missing the necessary 3 OH group for formation of a phosphodiester bond, this dntp cannot be incorporated. The dntp binding stabilizes the complex by decreasing the RT dissociation rate, thus trapping RT and the primer/template in a complex called dead end complex (DEC) 126,127. Any modification of the NRTI that increases the primer fraction in the nucleotide-binding site (N-site) increases the excision mechanism. This will probably explain why the excision reaction is relatively specific for AZT because its 3 OH group is replaced by a bulky azido group. 18

19 Discrimination K65R 69insert-complex# L74V V75T Q151M~ M184V/I all NRTIs, except AZT all NRTIs ddi, ddc, ABC d4t, ddi all NRTIs, except TDF 3TC, FTC, ddc, ABC General introduction Table 1. Overview of the major mutations in RT conferring resistance to NRTIs and their mechanism of resistance. * TAMs (thymidine associated mutations); usually two or more mutations are necessary to confer (high level) resistance. " TAMs are selected primarily by thymidine analogues, but confer resistance to other NRTIs as well. # complex with at least M41L, L210W or T215Y/F. ~ complex with A62V, V75I, F77L and F116Y. ^ excision is only determined with PPi and not with ATP as phosphate donor. NRTI = nucleos/tide reverse transcriptase inhibitor. AZT; Zidovudine, d4t; Stavudine, ddi; Didanosine, ddc; Zalcitabine, ABC; Excision M41L*" AZT, d4t D67N*" AZT, d4t 69insert-complex# all NRTIs K70R*" AZT, d4t V75T ^ d4t, ddi L210W*" AZT, d4t T215Y/F*" AZT, d4t K219Q/E*" AZT, d4t Abacavir, 3TC; Lamivudine, FTC; Emtricitabine, TDF; Tenofovir disoproxil fumarate. Several amino acid changes, such as K65R, L74V, L100I, A114S, Y181I/C and M184V, have been shown to diminish the excision reaction. Non-nucleoside reverse transcriptase inhibitor resistance Resistance to the other class of RTIs, the non-nucleoside inhibitors, is fairly different from NRTI-resistance. Due to their high specificity and common conformational properties, NNRTIs can easily select for mutant virus isolates. The most common NNRTI-resistance mutation is the K103N change, conferring resistance to EFV, NVP and DLV. The Y181C change and double mutant K103N+L100I are very prevalent as well. As shown in Figure 2 NNRTI-resistance can be accomplished by mutations at different codons in RT that line the NNRTIbinding pocket ; L100I, K103N, V106M/A, V108I, V179D, Y181C/I, Y188L/C/H, G190A/S/E, P225H and P236L 94. The K103N and Y181C changes are the most common substitutions conferring resistance to NNRTIs. The mechanism of resistance conferred by some of these substitutions has been studied in more detail 128,129. The amino acid change at position 103 is responsible for a network of hydrogen-bonds that may interfere with the ability of the NNRTIs to interact with its binding-site 128. The changes at codon 181 and 188 confer resistance by the loss of the aromatic ring-stacking interactions for the first-generation NNRTIs 129. Most NNRTI-resistance mutations confer resistance to all NNRTIs, resulting in a high level of cross-resistance among this class of RTIs. However, with respect to the P225H and P236L changes it has been suggested that these confer an increased sensitivity to certain NNRTIs, but the clinical significance needs to be further elucidated 130,

20 Chapter 1 Cross-resistance and synergy Of growing interest is the interplay between NRTIs, NNRTIs and/or resistanceassociated mutations. At first it was reported that AZT-resistant virus conferred no cross-resistance towards 3TC and nor did the M184V change confer resistance to AZT 117. Later, it was demonstrated that M184V increased susceptibility to AZT in wild type virus. Moreover the M184V change resensitizes TAM-harbouring viruses to AZT by inhibiting the excision of AZT 124,125,132,133. The precise mechanism is not completely clear but the valine alters the polymerase active site either by repositioning the primer/template complex and moves it from the ATP binding site or by increasing the fraction of AZT-terminated primer in the primer binding site (Psite) by relaxing steric constraints 124,134. The L74V change conferring resistance to didanosine (ddi), another nucleoside analogue is able to resensitize the TAM-mutated virus to AZT as well 135. Two reports have demonstrated that this amino acid substitution is able to reduce the ATP-dependent removal of AZT-MP, although the precise mechanism is not completely clear yet 136,137. Another example has been foscarnet (PFA). PFA is a simple PPi analogue and inhibits HIV-1 RT by competing with PPi for the PPi binding site, only when the primer is in the N-site. Several PFA-specific mutations can be selected during PFAtreatment of cytomegalovirus infections in HIV-infected patients, as well as the K65R. As described before, the K65R change confers resistance via the discrimination pathway to all approved NRTIs, but confers no resistance against AZT 138. By affecting the interaction with PPi, ATP and PFA this mutation not only creates a PFA-resistant RT but inhibits the unblocking reaction as well. Other studies have confirmed that the K65R mutation restores AZT sensitivity in TAMmutated viruses The same is true for the A114S substitution, which confers resistance to PFA. This change diminishes the enhanced pyrophosphorolysis activity and processivity associated with AZT-resistant RT 143. Besides these NRTIs and PFA, several non-nucleoside RT inhibitors have shown high-level synergy with AZT in inhibiting the emergence of AZT-resistant viruses. These NNRTIs, such as nevirapine, TIBO and the thiocarboxanilide NNRTI UC781, inhibit excision and resensitizes AZT-resistant polymerase to AZT-TP. Several mutations which are selected by NNRTIs, such as Y181I/C, L100I, have been shown to suppress AZT resistance by respectively decreasing the binding efficiency of the pyrophosphate donor ATP or increasing the discrimination 134, Also, protease inhibitor resistance-mutations were able to increase AZT susceptibility of a TAM-mutated virus

21 General introduction Furthermore, some NRTI-resistance mutations, such as T215Y, have been observed that increase NNRTI susceptibility 149. This hypersusceptibility has been demonstrated to confer an advantage when initiating an NNRTI-based regimen, but further research is warranted to determine the mechanism of NNRTI hypersusceptibility 150,151. Altogether, this indicates there is a large and complex interplay between the different drug classes and mutations. Replication capacity of RTI resistant HIV As described before, it is predicted that almost any virus clone which has a mutation compared to wild type will have a lower replication capacity. It is important to understand the difference of replication capacity in the presence or absence of drugs. In the absence of drugs, replication capacity will mainly be determined by the intrinsic replication capacity of a virus because resistance does not play a role. In contrast, in the presence of drugs, resistance is an important factor determining viral replication capacity. Multiple studies have demonstrated that mutations in reverse transcriptase are responsible for a decrease in replication capacity in the absence of antiretroviral treatment 12,46,62,65,69,70,101, This seems plausible, because if these changes conferred a replicative advantage they would be selected from the quasispecies. Unfortunately, there is no consensus on the optimal assay to measure in vitro replication capacity of HIV-1 variants. The use of multiple different assays, viral constructs and reference strains in the different studies published make comparison of the results very difficult. In a recent study by Cong et al. the effect of eleven resistance mutations in RT (M41L, K65R, D67N, K70R, L74V, K103N, Y181C, M184V, L210W, T215Y and K219Q) on replication capacity was determined using replication competition experiments 187. In agreement with previous reports all changes, except L210W, conferred a lower replication capacity compared to wild type. The degree of loss in replication capacity conferred by an individual mutation can vary between patient isolates and is not the same in all published studies. In general, the K65R, M184V and T215Y changes confer a very large decrease in replication capacity 59,176,187, Two studies reported an increase in replication capacity for the Q151M and Y181C changes, which was not found in other studies 62,153,155,193,194 Replication capacity of viruses harbouring more than one resistance mutation is studied in several settings. In general, all resistant virus isolates demonstrated a decrease in viral replication capacity in the absence of drugs 62,65,101,154,171,175,181,183,189,195. Also, viruses containing an insertion around amino acid 69 in combination with specific TAMs showed a dramatic reduction in replication 21

22 Chapter 1 capacity when cultured in the absence of drugs 70,108, However, one study reported the higher replication capacity of a 67N/70R/215Y/219Q-virus clone compared to wild type, but only in peripheral blood mononuclear cells (PBMC), stimulated with PHA and IL-2 on day 10 after infection 181. The negative effect on replication capacity is not limited to observations done in vitro. Several studies demonstrated the disappearance of RT mutations-containing viruses and replacement by wild type after treatment discontinuation, indicating the negative impact on viral replication capacity in vivo as well 66, , In some studies resistance-associated mutations seem to persist after treatment-interruption, but these mutations are not consistent in all studies that have been performed so far This indicates that reversion to wild type is dependent on several factors, like the availability of (archived) wild type virus, the presence of additional mutations that increase the viral replication capacity and/or host factors, which need to be elucidated. During antiretroviral therapy two types of mutations have been distinguished; primary resistance-conferring and secondary compensatory mutations 12. Primary amino acid changes confer resistance but have a negative influence on replication capacity. Secondary compensatory changes usually do not have an (increasing) effect on drug resistance but result in an increase in viral replication capacity. The concept of compensatory mutations has been extensively studied in protease inhibitor resistance that showed selection of compensatory amino acid changes in the cleavage sites (substrate of the viral protease) 12,217,218 However, the presence and/or impact of compensatory changes in the field of reverse transcriptase inhibitor resistance are still not completely clear. As described previously, secondary mutations in RT (M41L, D67N, L210W, K219Q/E) have been proposed that might increase viral fitness of primary mutations, but their main effect seems to be an increase in drug resistance. Genetic barrier to resistance With the introduction of highly active antiretroviral therapy (HAART) in 1996, the treatment of HIV-infections has been improved enormously 219. Monotherapy with a reverse transcriptase inhibitor resulted in the rapid selection of a resistant variant that was most likely present in the viral quasispecies. The genetic barrier to resistance, often defined as the number of mutations required to overcome drugselective pressure, is very low, because usually one or two nucleotide changes are sufficient for viral escape 92,95,220,221. Targeting HIV-1 by different classes of drugs (HAART) increases the genetic barrier; i.e. multiple mutations that are not present in the treatment-naïve quasispecies are required for viral escape. Counting the number of mutations that are required for viral escape gives an estimation of the 22

23 General introduction genetic barrier of a specific regimen. However, the threshold above which clinically relevant resistance develops will also be dependent on the level of pre-existing resistance and on the replication capacity and resistance level of baseline and intermediate variants 222. Scope of this thesis Although HAART has significantly improved the morbidity and mortality of the disease, HIV-1 is still able to escape current treatment regimens in some infected individuals. Furthermore, approximately 10% of newly diagnosed European patients are primarily infected with a viral strain harbouring at least one resistanceassociated mutation 223. Together with the fact that there is interplay between the different drugs and resistance mutations, it is obvious that the treatment of HIV-1 infections is very complex and there are still many questions that remain to be elucidated. The studies described in this thesis were performed to understand which factors drive the evolution of reverse transcriptase inhibitor resistant HIV-1 variants. To gain more insight into early virological failure, the influence of the genetic barrier to resistance was determined (chapter 2). Furthermore, we assessed the impact of baseline polymorphisms to gain more insight into the effect of mutations that are selected during RTI-containing treatment on replication capacity (chapter 3). Furthermore we studied the evolution of NRTI resistance in the presence of drugs; is NRTI-resistant HIV-1 able to increase its fitness by the selection of novel resistance-mutations and/or compensatory changes (chapter 4 and 5)? Finally we investigated the evolutionary pathways of an NRTI-resistant virus after transmission in the absence of drugs (chapter 6 and 7). References 1. Barre-Sinoussi, F. HIV as the cause of AIDS. Lancet 348, (1996) 2. Gallo, R.C. et al. Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science. 220, (1983) 3. Daar, E.S. et al. Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N.Engl.J Med. 324, (1991) 4. Borrow, P. et al. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol. 68, (1994) 5. Koup, R.A. et al. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol. 68, (1994) 6. Dewhurst, S.L. et al. Pathogenesis and treatment of HIV-1 infection: recent developments (Y2K update). Front Biosci. 5, D30-D49 (2000) 7. UNAIDS/WHO AIDS Epidemic Update. (2006) 8. Wright, S. The roles of mutation, inbreeding, crossbreeding, and selection in evolution. Proceedings of the Sixth International Congress on Genetics (1932) 9. Eigen, M. Viral quasispecies. Sci.Am. 269, (1993) 10. Domingo, E. et al. RNA virus fitness. Rev.Med.Virol. 7, (1997) 11. Domingo, E. Viruses at the edge of adaptation. Virology. 270, (2000) 23

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33 Chapter 2 Low genetic barrier caused by interplay between mutations explains early virological failure of a triple reverse transcriptase inhibitor regimen M.C.D.G. Huigen 1, L. de Graaf 1, M.J.M. Flink 1, J. van Lunzen 2, A. Pangerl 3, I. Reeb 3, R. Schuurman 1, C.A.B. Boucher 1 and M. Nijhuis 1 Manuscript in preparation 1 Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, the Netherlands 2 University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Bristol-Myers Squibb, Munich, Germany

34 Chapter 2 Abstract The triple reverse transcriptase inhibitor regimen consisting of tenofovir disoproxil fumarate (TDF), didanosine (ddi) and efavirenz (EFV) is a potential once daily regimen. Unfortunately, this regimen demonstrated an unexplained number of virological failures in some trials. We investigated whether a low genetic barrier to resistance caused by interplay between mutations could explain this high level of virological failure. Twelve antiretroviral-naïve HIV-1 patients that showed early virological failure in a trial with TDF, ddi and EFV were included. The virus population at failure was genotypically analysed. Furthermore, a clonal analysis was performed for all baseline samples, with a limit of detection for mutations of 5%. A wide variety of resistance profiles was observed. The most commonly observed genotype at failure was a unique combination of only two mutations: the ddi/tdf-resistance mutation L74V with the EFV-resistance mutation G190S/E. The L74V change has previously been shown to compensate for the negative effect of the G190E on replication capacity. The pre-existence of a resistance-mutation (V108I or V179D) as a 5% minority variant was found in some baseline viral populations. In conclusion, we suggest that early virological failure in patients on a triple ddi/tdf/efv regimen can be explained by the interplay between two mutations. One change at position 190 causes resistance to the NNRTI but at the same time a severe reduction in replication capacity. The other change at position 74 causes resistance to the two nucleos/tides analogues in the regimen (ddi and TDF) and at the same time mitigates most of the effect of the G190E change on replication capacity. Due to this interplay, only two mutations will generate a virus resistant to all three components of this regimen with a replication capacity that is not severely compromised. Introduction Highly active antiretroviral treatment (HAART) of HIV-1 infections has significantly improved both the morbidity and mortality of the disease 1. Unfortunately, transmission or emergence of drug resistant viruses can be the cause of treatment failure. The genetic barrier of a specific regimen is considered an important determining factor for the development of resistance and duration of treatment response 2,3. This factor can be described as the difficulty for HIV to escape the antiretroviral drug pressure and is usually defined by the number of mutations required to overcome drug-selective pressure. For instance, the currently approved 34

35 Low genetic barrier explains early virological failure non-nucleoside reverse transcriptase inhibitors (NNRTIs) have a low genetic barrier when used as monotherapy since usually one nucleotide change is sufficient for viral escape 4. Development of novel HIV-1 inhibitors is largely focused on obtaining a high intrinsic genetic barrier to resistance. Good examples in this regard are the two new protease inhibitors (PIs) TMC114 (Darunavir) and PL-100 and the novel NNRTI TMC125 with an increased intrinsic genetic barrier 5-7. Furthermore ritonavir-boosting of PIs raises the (intracellular) drug levels and thereby increases the genetic barrier 8. Resistance development to (some of) these novel inhibitors, especially when used in combination in vivo, is very difficult because it requires more amino acid changes than are present in the wild type viral quasispecies. Estimations of the genetic barrier are performed by counting the number of mutations that are required for viral escape. However, the threshold above which clinically significant resistance develops is not only dependent on the number of mutations but also on the level of pre-existing resistance and the replication rate of these baseline variants which might influence the likelihood of occurrence 2. Given the relatively high genetic barrier, current HAART is successful but a major drawback is the increasing risk of toxicity and adverse side effects, such as lipodystrophy, diarrhoea, nausea and vomiting. Furthermore, it appears to be difficult to adhere to complex dosing schemes with often high pill burdens. Prescribing a combination of three reverse transcriptase (RT) inhibitors such as the NRTIs tenofovir disoproxil fumarate (TDF), didanosine (ddi) and the NNRTI efavirenz (EFV) has both the advantage of being a once daily regimen and saves the other classes of HIV-1 inhibitors for future therapy. This regimen showed to be effective as maintenance therapy, but unfortunately, early virological therapy failure was observed in a significant proportion of treatment-naïve patients Treatment failure could not be attributed to efavirenz exposure, poor adherence, pharmacological interactions or measurable baseline resistance. In the present study we determined if the frequent occurrence of early virological failure could be attributed to a low genetic barrier for the described regimen. Furthermore, we determined if the genetic barrier can be lowered by the presence of resistant variants as a minority population in the viral quasispecies before the onset of treatment, using a clonal sequence approach detecting minority variants representing 5% of the population. 35

36 Chapter 2 Results Genotypic analysis of patients on a failing ddi/tdf/efv regimen In Table 1 all amino acid changes that were observed during treatment failure of ddi/tdf/efv are indicated, representing a wide variety of resistance-profiles. The majority of patients harboured the NRTI-resistance mutation L74V/I and/or K65R, whereas three patients (9, 10 and 11) presented solely NNRTI-mutations upon treatment failure. The most commonly observed NNRTI resistance mutation was the G190E/S often combined with L74V/I, which was found in five of the eleven genotypes. Together, in most patients, therapy failure was found to be associated with the rapid selection of just one or two drug resistance associated amino acid changes in the viral RT enzyme, indicative of a low genetic barrier for this regimen. Table 1. Amino acid changes determination at early virological failure. resistance-associated with NRTIs NNRTIs amino acid in RT consensus B K L A L K K V V V V Y Y G P patient 1 week 13 L/V E patient 2 week 8 L/V E patient 3 week 24 V A/G Q/E patient 4 week 4 L week 12 L/V S patient 5 week 8 C/S week 14 K/R L/V E/K C/S patient 6 week 31 R patient 7 week 4 N H week 8 N H week 14 R I N patient 8 week 8 N I week 16 I/L N I H patient 9 week 8 D/V L patient 10 week 4 L I Y/C C/Y G/S week 8 N V/L I Y/C S/G patient 11 week 4 I/L K/T I/V D E/G patient 12 week 36 could not be determined Minority variant analysis of baseline quasispecies All twelve patients included in this study were treatment-naïve and did not reveal resistance-associated mutations at baseline using a population-based sequencing approach, except for the patient 10-derived virus that harboured a V118I change, which remained present during treatment. This amino acid change does not confer resistance on its own but most likely confers resistance when present in combination with thymidine analogue associated mutations To determine if drug-resistant minority variants were present in the viral population before start of treatment, the pre-therapy viral quasispecies of twelve patients that failed on the triple RTI regimen were analysed by genotyping twenty 36

37 Low genetic barrier explains early virological failure individual viral clones for each sample (Table 2). Whereas the routinely performed population-based sequencing can only reveal resistant HIV-1 variants if they are present in > 25% of the viral quasispecies, in the current approach detection of a variant in 5% of the population is possible. Of the twelve individuals failing on the described regimen, two patients (8 and 12) revealed the presence of the V108I amino acid change in 5% of the viral quasispecies (1 of 20 viral clones). Also, the viral pretreatment population of patient 12 showed the presence of the T69N in one of the twenty viral clones. This amino acid change is associated with NRTI treatment, but its effect on NRTI susceptibility is unknown 17 (Stanford HIV drug resistance database). Another patient (11) revealed the presence of the V179D change in 1 out of 20 clones within the viral population. Although this change is not listed by the International AIDS Society (IAS) it has been shown to be associated with resistance to NRTIs or NNRTIs 18 (Stanford db). Finally, we noticed that the remaining eight patients (1-7 and 9) did not reveal evidence of the presence of a minority resistant virus in 5% (or more) in the viral quasispecies before onset of treatment. 37

38 Chapter 2 Table 2. Analysis of the baseline viral quasispecies. The viral quasispecies of twelve patients failing on the triple TDF/ddI/EFV regimen was investigated by the analysis of the N-terminal part of RT of twenty individual viral clones. Shown are the amino acid changes compared to consensus B of the population sequence before onset of treatment, the twenty viral clones and if available the genotype at virological failure. Patient 1 amino acid in RT genotype consensus B T V T L K D R T I N V I G I E Q R V K K T P population baseline I E V Q/H K T Q A T clonal clone 1 I E V K T Q A T clonal clone 2 I E V H K T Q A T clonal clone 3 I E V K T Q A T clonal clone 4 I E V K T Q A T clonal clone 5 I E V K T Q A S clonal clone 6 I E V H K T Q A T clonal clone 7 I E V H K T Q A T clonal clone 8 I E V Y V K T Q A T clonal clone 9 I E V H K T Q A T clonal clone 10 I E V H K T Q A T clonal clone 11 I E V K T Q R A T clonal clone 12 I E V H K T Q A T clonal clone 13 I E G V H K T Q A T clonal clone 14 I E V M H K T Q A T clonal clone 15 I E V K T Q A T clonal clone 16 I E V V K T Q A S clonal clone 17 I E V H K T Q A T clonal clone 18 I E V H K T Q A T clonal clone 19 I E V K T Q A T clonal clone 20 I E V K T Q A T population week 4 T/A I E V K T Q A T population week 13 I N L/V E E K V E E/G K T Q A T Patient 2 amino acid in RT genotype consensus B K V K E L D I V G I E Q R I T population baseline R I D T V K/E K A A clonal clone 1 R I D T V K A A clonal clone 2 R I D T V K A A clonal clone 3 R I T D T V K K A A clonal clone 4 I E T V K A A clonal clone 5 R I D T V K A A clonal clone 6 R I D T V K K A A clonal clone 7 R I D T V K A A clonal clone 8 R I D T V K A A clonal clone 9 R I D T V K A A clonal clone 10 R I D T V K A A clonal clone 11 R I D T V K A A clonal clone 12 R I D T V K A N A clonal clone 13 I E T V K A A clonal clone 14 R I D T V K A A clonal clone 15 R I D F T V K A A clonal clone 16 R I D T V K K A A clonal clone 17 R I D T V K K A A clonal clone 18 R I D T V K A A clonal clone 19 R I E T V K A A clonal clone 20 R I D T V K A A population week 8 K/R I L/V T G/V E I/V K/Q A A Patient 3 amino acid in RT Patient 4 amino acid in RT genotype consensus B P T I L R A K D S F G T E Q R K H V E D V A K Q T genotype consensus B E I L R P T V D T D Y G T R D E V W A I T E population baseline K E A/C I G K E E/D P/A R Q/H A population baseline K V K E P V S K clonal clone 1 K E A I G K P R clonal clone 1 K A V K E P V S K clonal clone 2 S K E A I E K E D P R H clonal clone 2 K A V K E P V K clonal clone 3 S K E A I E K E E P R H A clonal clone 3 K V K E P S K clonal clone 4 S K E C I E K E P R H A clonal clone 4 K V K E P V S K clonal clone 5 K E C I G K E R A clonal clone 5 K V K E P S K clonal clone 6 K E C I G K E R H A clonal clone 6 K A V K E P V K clonal clone 7 K E C I G K E E R A clonal clone 7 K I K E P V S K clonal clone 8 K E C I G K E P R H clonal clone 8 K V K E P S K clonal clone 9 K E E C I G K E D R H A clonal clone 9 K V K E P S K clonal clone 10 K E T I I G K L E D R A clonal clone 10 G K G I K E P S K clonal clone 11 K E C I G K E R A clonal clone 11 K S I K E P S K clonal clone 12 K V K N E P S K clonal clone 12 K E C I G K E E P R H A clonal clone 13 K V K E P V S K clonal clone 13 K E A I K G K E E P R H clonal clone 14 K V K E P V K clonal clone 14 S K E C I E K E E P R H A clonal clone 15 K I K E P V P K clonal clone 15 K E A I G K E R A clonal clone 16 K M V K E P S K clonal clone 16 K E C I E K E E R A clonal clone 17 V K I K E P V S K clonal clone 17 K E C I G K E R H A clonal clone 18 K I K E P V K clonal clone 18 I K E C I G K E R A clonal clone 19 K I K E * P S K clonal clone 19 K E A I G K E R H A clonal clone 20 K I k E P V S K clonal clone 20 F K E C I G K E E A P R H A population week 4 K L V K K E P S K population week 24 V K A/G E A Q/E I K G K K/N E P R H A population week 12 L/V K D/N S I/V K E P I/V S K clone 1: additional Van91I restriction site around codon 225 * stopcodon 38

39 Low genetic barrier explains early virological failure Table 2. Continued. Analysis of the baseline viral quasispecies. Patient 5 amino acid in RT genotype consensus B E A K K L K A I I Q I T K V V G T I L V D D A I population baseline V V I/V T/I A F M I/V clonal clone 1 R T V I F M clonal clone 2 V V V E A F M V clonal clone 3 K V V V A F M V clonal clone 4 V V V A F M V clonal clone 5 V V R V A F M V clonal clone 6 V I I F M clonal clone 7 V V V A F M V clonal clone 8 V V V A F M clonal clone 9 V V V A F M V clonal clone 10 V I V F M clonal clone 11 V V V V A F M Y V clonal clone 12 V V V A F M G T V clonal clone 13 V V V A F M V clonal clone 14 V V V A F M V clonal clone 15 V V V A F M V clonal clone 16 V V V A F M V clonal clone 17 V I F M clonal clone 18 V V V A F M V clonal clone 19 V V V I A F M V clonal clone 20 V I A F M population week 4 I/V V I/V T/I A F M population week 8 V V V C/S A F M resistance mutations at week 14 K/R L/V E/K C/S Patient 7 amino acid in RT genotype consensus B A V K K K T R W L K K Y S D T V L G S K N P Q T R P T V L S K V L K I P population pre screen I/V K E K/N K Q I I V Q clonal clone 1 K E K K G Q I I V Q clonal clone 2 R K E K K Q I I V Q clonal clone 3 I K E E K K Q I I V Q clonal clone 4 K E N K Q I I V Q clonal clone 5 K E K K I Q I I V Q clonal clone 6 I K H E K K Q I I V Q clonal clone 7 I K G E N K Q I I V Q clonal clone 8 K E N K Q I I V Q clonal clone 9 K L V E N K Q I I V Q clonal clone 10 K E K K S Q I I V Q clonal clone 11 T K Q E N K Q I I V Q clonal clone 12 I I K F E N K Q I I V Q clonal clone 13 K E K K Q I I V Q clonal clone 14 K E K K Q I I V Q clonal clone 15 K E K K M Q I I R V Q clonal clone 16 # K P S E K K Q I I V Q clonal clone 17 R K A E N K Q I I V Q clonal clone 18 K * I E K K Q I I V Q clonal clone 19 K E N K Q I I V Q clonal clone 20 K E N K Q I I V Q population week 4 K N E K K H Q I I V Q population week 8 K K/R N E K K H Q I I V Q population week 14 R K I N E K K Q I I V Q # 1 nucleotide deletion at aa 101 * stopcodon Patient 6 amino acid in RT genotype consensus B V T K S K K K V F K I I Q S K Q D V population baseline T I G Q/R K/R E V A S R E V/I clonal clone 1 T I G Q R E V A S R E I clonal clone 2 T I G Q R E V A S R E I clonal clone 3 T I G Q R E V A S R E I clonal clone 4 T I G Q R E V A S R E I clonal clone 5 T I G Q R E V A S R E clonal clone 6 T I G Q R E V A S R E clonal clone 7 T I G Q R E V A S R E clonal clone 8 T I G Q R E V A S R E I clonal clone 9 T I G R I E V A S R E clonal clone 10 T I G Q R E V A S R E clonal clone 11 T I G Q R L E V V A S R E clonal clone 12 T V Q E V A S R E clonal clone 13 T I G Q R E V T A S R E clonal clone 14 T V G R E V R A S R E clonal clone 15 T I G Q R E V A S R E clonal clone 16 T I G R R E V A S R E clonal clone 17 T I G Q R E V A S R E I clonal clone 18 T I G Q R E V A S R E I clonal clone 19 T I G Q R E V T A S R E clonal clone 20 T I G Q R E V A S R E resistance mutations at week 31 R silent amino acid change at codon 181 causes additional Van91I restriction site Patient 8 amino acid in RT genotype consensus B E V E V K D L K V D I E T S T K D V K Q R P F V I population screen E C E K S clonal clone 1 K I E C E K S clonal clone 2 E C E K S clonal clone 3 E C E E K S clonal clone 4 E C E K S clonal clone 5 E C E K S clonal clone 6# L R E C E K S clonal clone 7 L G E C E K S clonal clone 8 E C E K S clonal clone 9 I E C E K S M clonal clone 10 E C E E K S clonal clone 11 E G C E K S clonal clone 12 E C E K S clonal clone 13 L E C E K S clonal clone 14 E C E K S clonal clone 15 E C E K S clonal clone 16 I E C E E K S clonal clone 17 E C A E E K S clonal clone 18 E C E K S S clonal clone 19 E C E I K S S clonal clone 20 E C E K S population week 4 E C E K/R K S M/V population week 8 N I E T A C E K S population week 16 E/K I/L N I E T A C E K S H # 1 nucleotide deletion at aa

40 Chapter 2 Table 2. Continued. Analysis of the baseline viral quasispecies. Patient 9 amino acid in RT genotype consensus B E V I E D K F T S I V Y G Q T R P D E L V V Y K K T K population baseline Y E C E K/R K/R T K/Q R A/T clonal clone 1 G Y E C E R K T R A clonal clone 2 Y E C E K I K T H R A R clonal clone 3 Y E C E R K T R A clonal clone 4 Y E C V E K K T Q R A clonal clone 5 Y E C E K L T I Q R A clonal clone 6 Y E C E R K T Q R A clonal clone 7 Y E C E K K T R A R clonal clone 8 K T Y E C E K K T Q R clonal clone 9 Y E C E K N K T R A clonal clone 10 Y E S Y E K K T Q R A clonal clone 11 Y E C E R K T R A clonal clone 12 Y E C E K K T Q R A clonal clone 13 Y E C E K K T Q R A clonal clone 14 Y E A C I E K K K T Q R A clonal clone 15 Y E C E K T Q R A clonal clone 16 Y E C E R K T R clonal clone 17 Y E C E K K T R A clonal clone 18 Y E R E R K T R A clonal clone 19 Y E C E K K T R A clonal clone 20 Y E C E R K T R A population week 4 L Y E C E K K T R A population week 8 Y E C D/V L E K K L/I T Q R A Patient 10 amino acid in RT genotype consensus B V G I R L K V A V S K D I T Y Y G T I E Q R F L V G A K L L T I P population baseline I I E E M I A/V E/G K L P R A V clonal clone 1 I I E E M I A E K L M P R A V clonal clone 2 I E I E E M I A E K L P R A V clonal clone 3 I K I E E M I A E K L E P R A V clonal clone 4 I T I E E M I V E K L M P R A V clonal clone 5 I E I E E M I A E K L E P R A V clonal clone 6 I F I E E M I A E K L P R A V clonal clone 7 I I E E M I A E K L E P R I I V clonal clone 8 I I E E M I A * E K L M P R A V clonal clone 9 I I E E M I V E K L P R I V clonal clone 10 I I E E M I A G K L P R A V clonal clone 11 I E I E E M I A E K L P R A V clonal clone 12 I I E E M I A E K L P R V clonal clone 13 I I E E I A E K L P R V clonal clone 14 I I E E M I A E K L E P R A V clonal clone 15 I I E M I A V G K L P R A V clonal clone 16 I I E E M I A E K L F P R A V A clonal clone 17 I I E E M I A E K L P A V clonal clone 18 I N I E E M I A E K L P R A V clonal clone 19 I I S E M I A E K L P A V clonal clone 20 I I E E M I A E K L P R I V population week 4 I L I E E M I Y/C C/Y G/S A E K L P R A V population week 8 I N V/L I E E M/I I Y/C S/G A E K L R/L E/V P K/R A V * stopcodon Patient 11 amino acid in RT genotype consensus B V T E K E K L K K V L K D I S D V G T E Q R H T V V A T T I E population baseline I A/T E/D E/D E E L E A E P A V clonal clone 1 I A D E E L E A E P A V clonal clone 2 I A D E E L E A E I P A V clonal clone 3 I A D E E L E A E P A V clonal clone 4 I A E E L E A E P A V clonal clone 5 I A E E L E A E I P A V clonal clone 6 I A R D E E L E A E K P A V clonal clone 7 I A D E E L E A E P A V clonal clone 8 I D R E L E D E P A V clonal clone 9 I A D E E L E A E P A V V clonal clone 10* I A D E E L E A I E K P A A clonal clone 11 I A E E L E A K E P A V clonal clone 12^ I A D R E E A E P A V clonal clone 13 I A D E E L E A E P A V clonal clone 14^ I A E A E P A V clonal clone 15 I A E E L E A E P A V clonal clone 16* I A D E E L E A A E P A V clonal clone 17 I A D E E L E A E P A V clonal clone 18 I A R E E L E A E P A A V V clonal clone 19 I A D E E L E A E P A V clonal clone 20 I D E E L N E A E P A V population week 4 I D/E I/L K/T I/V E E L E D E/G E H/Y I/V P A V * 1 nucleotide deletion ^ deletion aa Patient 12 amino acid in RT genotype consensus B E T R V G K I V K I P population baseline E T K R V clonal clone 1 E T K R V clonal clone 2 E T K R V clonal clone 3 E T K R V clonal clone 4 E T K M V clonal clone 5 E T K R V clonal clone 6 E T K R V clonal clone 7 D K S E T K R V clonal clone 8 E T K R V clonal clone 9 E T K R V clonal clone 10 D K E T K R V clonal clone 11 E T K R V clonal clone 12 E T K R V clonal clone 13 E T K R V clonal clone 14 N E T K R V S clonal clone 15 D E T K R V clonal clone 16 E T K R V clonal clone 17 E T K R V clonal clone 18 E T K R V clonal clone 19 I E T K R V clonal clone 20 D K E T K R V failure week 36 could not be determined clone 1, 11 and 16 insertion of 1 nucleotide at position 31 40

41 Low genetic barrier explains early virological failure Discussion Emergence of drug-resistance and complexity of the dosing regimen coupled with poor adherence and drug toxicities are major limitations of the current treatment of HIV-1 infections. Several approaches are undertaken to simplify and optimize current therapies. Unfortunately, a once daily regimen of solely RTIs (ddi/tdf/efv) revealed unexplained early virological failure in several treatmentnaïve patients In this study it was shown that two nucleotide changes were sufficient for viral escape in several patients, indicating the low genetic barrier of this regimen. It has been suggested that the failure of triple NRTI regimens such as lamivudine, tenofovir and abacavir or didanosine is attributed to their low genetic barrier (M184V and/or K65R) The genetic barrier cannot be simply defined by the number of escape mutations compared to the baseline population sequence 19,23. The level of preexisting resistance and replication capacity of (intermediate) resistant variants will influence the genetic barrier to resistance as well. The influence of replication capacity might be demonstrated by an interesting combination of L74V and G190S/E that was noticed in several patients that were included in our study. Previous in vitro analyses have shown that two specific classes of NNRTIs (quinoxaline derivatives and (alkylamino)piperidine bis(heteroaryl)piperizine (AAP- BHAP)) selected for the G190E change, which conferred resistance to these NNRTIs The selection of this change resulted in a dramatic reduction in replication capacity, caused by large reductions in the polymerase and RNase H specific enzymatic activities 27. The G190E change showed cross-resistance to both nevirapine and EFV. Subsequent research showed that during continued NNRTI pressure, the G190E-virus acquired additional changes at position 74 (L74V/I) or 75 (V75L/I) in RT 28. The V75I or L74V change appeared to be compensatory and mitigated the deleterious effect of the G190E change on RT function 29. The L74V confers resistance to ddi and TDF and will therefore have two functions: reducing the susceptibility for ddi and TDF and increasing the replication capacity. Thus, the interaction between these changes will generate a fully resistant virus with a replication capacity that is not severely compromised but even compensated. Furthermore, this indicates that a genetic barrier of two mutations is too low for effective antiretroviral treatment, which confirms previous studies Also, pre-existing mutants existing as an undetected minority population will lower the genetic barrier to resistance. Indeed, in this study in several patients the pre-existence of a drug-resistant minority variant was demonstrated. This is in line 41

42 Chapter 2 with other studies that also showed the presence of drug-resistant minority variants in the viral population of antiretroviral therapy naïve patients 30,31. Furthermore, it has been demonstrated that pretherapy presence of minority drug-resistant variants that remain undetected using standard genotyping, may compromise treatment efficacy We speculate that the presence of minority drug-resistant variants at baseline determines the evolutionary pathway of resistance. This may explain why not all viruses have selected the L74V and G190E/S combination. Given that more sensitive genotyping techniques have been developed in the recent years, the interest in minority populations and their effect on treatment efficacy is increasing. An important point of debate is what percentage of a specific resistance-associated mutation in the viral population is clinically significant. HIV-1 replicates as a viral quasipecies and it is estimated that every single nucleotide mutation is being generated every day. This means that with a very sensitive assay you are bound to find a particular resistant variant in generally all viral populations. Our clonal analysis has a detection limit of more than 5% which is relatively high. To date, no consensus is available on the clinical significant or relevant percentage of a specific virus in the population. A potential limitation of the clonal analysis is that we cannot exclude that the mutations are being generated during every PCR-amplification, but the chance that the assay-introduced nucleotide change is exactly the substitution that is selected in the patients failing treatment seems neglectable small. Several recent reports have identified novel amino acid changes that are associated with RTI-resistance Although their clinical significance is not fully elucidated yet, we speculate that the presence of such polymorphisms at baseline may influence the treatment efficacy in patients failing treatment. In our study population we found several of these amino acid changes that are associated with RTI-resistance (including ddi, TDF and EFV), that were either selected or persisted in the population during treatment (V60I, K122E, I135T/V, G196E, T200A/I/K, E203K, Q207E, R211K/S and L283I). However, in a few patients failing treatment some of these changes were not selected from the viral quasispecies, indicating that there is complex interplay between different amino acid changes that are responsible for selection during treatment. Further research is warranted to elucidate the clinical significance of these novel mutations. In conclusion, we suggest that early virological failure in patients on a triple ddi/tdf/efv regimen might be explained by its low genetic barrier. Escape with two interacting mutations (L74V and G190E) and the presence of minority variants before treatment will lower the genetic barrier and influence treatment efficacy, but needs further investigation. 42

43 Low genetic barrier explains early virological failure Materials and Methods RNA isolation HIV-1 nucleic acids were isolated according to the method described by Boom et al. 42. Briefly, samples were resuspended in lysis buffer containing silica (NucliSens, Organon Teknika, Boxtel, the Netherlands) and incubated for 10 minutes at room temperature to let the nucleic acids bind to the silica. The samples were washed twice with washing buffer (NucliSens), twice with ethanol and once with acetone (Merck) before the nucleic acids were eluted in 100 µl poly (A) RNA (40ng/µl). Generation of 20 individual clones representing the viral quasispecies The amplified N-terminal part of reverse transcriptase was used to generate recombinant virus clones containing amino acid 25 till 314 from RT in a wild type (HXB2) backbone as described previously 43. Briefly, the amplified N-terminal part of RT was digested with 1 Unit Mlu NI (Roche) and 1 Unit Van91 I (Roche) and subsequently ligated O/N at 4 ºC with a digested HXB2-vector lacking the N-terminus RT (amino acid ) using T4 ligase (Promega). To prevent religation of the vector the ligation product was digested with 1 Unit Asp I (Roche) before transformation into E.Coli JM109 High Efficiency Competent Cells (Promega) using a heat-shock at 42ºC. The transformed cells were cultured for 1 hour in Luria-Bertani (LB)-medium at 37ºC and plated out on an LB-agar plate containing 40 µg/ml ampicillin (Clamoxyl, GlaxoSmith Kline BV). Single colonies containing plasmid representing one RT gene were picked and cultured overnight at 37 C in 5 ml LB- medium supplemented with ampicillin (40 ug/ml). The plasmid was isolated using the QIAprep spin Miniprep kit (Qiagen) and genotypically analyzed using (a subset of) primers RT19, RT20, BRT, BRRT and KRT as described previously 43. To determine the number of nucleotide changes that are generated in this amplification protocol due to errors introduced in the cdna synthesis and amplification, the complete procedure was performed with a wild type virus clone. Twenty individual clones were genotypically analysed and revealed an average of 1 random nucleotide change per virus clone compared to the original sequence (18 substitutions in 20 clones). Thus, an average of one random nucleotide change per 1000 basepairs must be attributed to the amplification procedure. Sequence analysis was performed using the BigDye Terminator v1.1 or v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI3100 Genetic Analyzer. Acknowledgements This study was financially supported by unrestricted institutional grant from Bristol-Myers Squibb. References 1. Palella, F.J., Jr. et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N.Engl.J.Med. 338, (1998) 2. Beerenwinkel, N. et al. Estimating HIV evolutionary pathways and the genetic barrier to drug resistance. J.Infect.Dis. 191, (2005) 3. van de Vijver, D.A. et al. The calculated genetic barrier for antiretroviral drug resistance substitutions is largely similar for different HIV-1 subtypes. J.Acquir.Immune.Defic.Syndr. 41, (2006) 4. Zhang, Z. et al. Clinical utility of current NNRTIs and perspectives of new agents in this class under development. Antivir.Chem.Chemother. 15, (2004) 5. Koh, Y. et al. Novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI) UIC (TMC114) with potent activity against multi-pi-resistant human immunodeficiency virus in vitro. Antimicrob.Agents Chemother. 47, (2003) 43

44 Chapter 2 6. Wu, J.J. et al. The HIV-1 protease inhibitor PL-100 has a high genetic barrier and selects a novel pattern of mutations. Antiviral Therapy 11, S152 (2006) 7. Vingerhoets, J. et al. TMC125 displays a high genetic barrier to the development of resistance: evidence from in vitro selection experiments. J Virol. 79, (2005) 8. Kempf, D.J. et al. Pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus protease by coadministration with ritonavir. Antimicrob.Agents Chemother. 41, (1997) 9. Barrios, A. et al. Simplification therapy with once-daily didanosine, tenofovir and efavirenz in HIV-1-infected adults with viral suppression receiving a more complex antiretroviral regimen: final results of the EFADITE trial. Antivir.Ther. 10, (2005) 10. Maitland, D. et al. Early virologic failure in HIV-1 infected subjects on didanosine/tenofovir/efavirenz: 12-week results from a randomized trial. AIDS. 19, (2005) 11. Van Lunzen, J. et al. High Rate of Virological Failure during Once-daily Therapy with Tenofovir + Didanosine 250mg + Efavirenz in Treatment-naive Patients. IAS Conf. HIV Pathog. Treat abstract TuPp0306 (2005) 12. Torti, C. et al. Early virological failure after tenofovir + didanosine + efavirenz combination in HIV-positive patients upon starting antiretroviral therapy. Antivir.Ther. 10, (2005) 13. Podzamczer, D. et al. Early virological failure with a combination of tenofovir, didanosine and efavirenz. Antivir.Ther. 10, (2005) 14. Montes, B. & Segondy, M. Prevalence of the mutational pattern E44D/A and/or V118I in the reverse transcriptase (RT) gene of HIV-1 in relation to treatment with nucleoside analogue RT inhibitors. J Med.Virol. 66, (2002) 15. Girouard, M. et al. Mutations E44D and V118I in the reverse transcriptase of HIV-1 play distinct mechanistic roles in dual resistance to AZT and 3TC. J Biol.Chem. 278, (2003) 16. Hertogs, K. et al. A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. Antimicrob.Agents Chemother. 44, (2000) 17. Winters, M.A. & Merigan, T.C. Variants other than aspartic acid at codon 69 of the human immunodeficiency virus type 1 reverse transcriptase gene affect susceptibility to nuleoside analogs. Antimicrob.Agents Chemother. 45, (2001) 18. Parkin, N.T. et al. The K101P and K103R/V179D mutations in human immunodeficiency virus type 1 reverse transcriptase confer resistance to nonnucleoside reverse transcriptase inhibitors. Antimicrob.Agents Chemother. 50, (2006) 19. Luber, A.D. Genetic barriers to resistance and impact on clinical response. MedGenMed. 7, 69 (2005) 20. Gallant, J.E. et al. Early virologic nonresponse to tenofovir, abacavir, and lamivudine in HIVinfected antiretroviral-naive subjects. J.Infect.Dis. 192, (2005) 21. Ruane, P.J. & Luber, A.D. K65R-associated virologic failure in HIV-infected patients receiving tenofovir-containing triple nucleoside/nucleotide reverse transcriptase inhibitor regimens. MedGenMed. 6, 31 (2004) 22. Landman, R. et al. Early virologic failure and rescue therapy of tenofovir, abacavir, and lamivudine for initial treatment of HIV-1 infection: TONUS study. HIV.Clin.Trials 6, (2005) 23. Gallant, J.E. et al. Efficacy and safety of tenofovir DF vs stavudine in combination therapy in antiretroviral-naive patients: a 3-year randomized trial. JAMA 292, (2004) 24. Kleim, J.P. et al. Activity of a novel quinoxaline derivative against human immunodeficiency virus type 1 reverse transcriptase and viral replication. Antimicrob.Agents Chemother. 37, (1993) 25. Kleim, J.P. et al. Mutational analysis of residue 190 of human immunodeficiency virus type 1 reverse transcriptase. Virology. 200, (1994) 26. Olmsted, R.A. et al. (Alkylamino) piperidine bis(heteroaryl)piperizine analogs are potent, broadspectrum nonnucleoside reverse transcriptase inhibitors of drug-resistant isolates of human immunodeficiency virus type 1 (HIV-1) and select for drug-resistant variants of HIV-1IIIB with reduced replication phenotypes. J Virol. 70, (1996) 27. Fan, N. et al. A drug resistance mutation in the inhibitor binding pocket of human immunodeficiency virus type 1 reverse transcriptase impairs DNA synthesis and RNA degradation. Biochemistry. 35, (1996) 28. Kleim, J.P. et al. Selective pressure of a quinoxaline nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on HIV-1 replication results in 44

45 Low genetic barrier explains early virological failure the emergence of nucleoside RT-inhibitor-specific (RT Leu-74-->Val or Ile and Val-75-->Leu or Ile) HIV-1 mutants. Proc.Natl.Acad.Sci.U.S.A 93, (1996) 29. Boyer, P.L. et al. A mutation at position 190 of human immunodeficiency virus type 1 reverse transcriptase interacts with mutations at positions 74 and 75 via the template primer. Antimicrob.Agents Chemother. 42, (1998) 30. Metzner, K.J. et al. Detection of minor populations of drug-resistant HIV-1 in acute seroconverters. AIDS 19, (2005) 31. Johnson, J. et al. Baseline detection of low-frequency drug resistance-associated mutations is strongly associated with virological failure in previously antiretroviral naive HIV-1-infected persons. Antiviral Therapy 11, S69 (2006) 32. Van Laethem, K. et al. No response to first-line tenofovir+lamivudine+efavirenz despite optimization according to baseline resistance testing: Impact of resistant minority variants on efficacy of low genetic barrier drugs. J Clin Virol39, (2007) 33. Briones, C. et al. Minority memory genomes can influence the evolution of HIV-1 quasispecies in vivo. Gene 384, (2006) 34. Mellors, J. et al. Low-frequency NNRTI-resistant Variants Contribute to Failure of Efavirenzcontaining Regimens. CROI-abstract 39 (2004) 35. Perno, C.F. et al. Novel drug resistance mutations in HIV: recognition and clinical relevance. AIDS Rev. 8, (2006) 36. Svicher, V. et al. Involvement of novel human immunodeficiency virus type 1 reverse transcriptase mutations in the regulation of resistance to nucleoside inhibitors. J.Virol. 80, (2006) 37. Ceccherini-Silberstein, F. et al. High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations. J.Virol. 79, (2005) 38. Saracino, A. et al. Impact of unreported HIV-1 reverse transcriptase mutations on phenotypic resistance to nucleoside and non-nucleoside inhibitors. J Med.Virol. 78, 9-17 (2006) 39. Cane, P.A. et al. Identification of accessory mutations associated with high-level resistance in HIV-1 reverse transcriptase. AIDS 21, (2007) 40. Gonzales, M.J. et al. Extended spectrum of HIV-1 reverse transcriptase mutations in patients receiving multiple nucleoside analog inhibitors. AIDS 17, (2003) 41. Rhee, S.Y. et al. Human immunodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Res. 31, (2003) 42. Boom, R. et al. Rapid and simple method for purification of nucleic acids. J.Clin.Microbiol. 28, (1990) 43. van Maarseveen, N.M. et al. A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants. J Virol.Methods 133, (2006) 45

46 Chapter 2 46

47 Chapter 3 Evaluation of four approaches to measure replication capacity of reverse transcriptase inhibitor resistant HIV-1 variants M.C.D.G Huigen 1, M. Nijhuis 1, L. de Graaf 1, Y. Lie 2, A. de Ronde 3, D. Perez-Bercoff 4, J. Balzarini 5, H. Walter 6, S. Corvasce 7, D.K. Stammers 8, C. Cabrera 9, C. Balotta 7, F. Clavel 4, B. Berkhout 3, N.T. Parkin 2, C.A.B. Boucher 1 and R. Schuurman 1 Manuscript in preparation 1 Department of Medical Microbiology, University Medical Center Utrecht, the Netherlands, 2 Monogram Biosciences, South San Francisco, CA, USA 3 Laboratory of Experimental Virology, Department of Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, the Netherlands 4 Inserm U552, IMEA, Hopital Bichat Institut National de la Santé et de la Recherche Médicale, Paris, France 5 Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Katholieke Universiteit Leuven, Belgium 6 National Reference Centre for Retroviruses,Institute for Clinical and Molecular Virology, Erlangen, Germany 7 Department of Clinical Sciences "Luigi Sacco", Section of Infectious Diseases and Immunopathology, University of Milan, Milan, Italy 8 The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, University of Oxford, United Kingdom 9 Department of Retrovirology, Hospital Universitari Germans Trias i Pujol, Badalona, Spain

48 Chapter 3 Abstract The effect of resistance-associated mutations on replication capacity (RC) was investigated in a set of baseline and failure samples derived from fifteen patients on a non-suppressive reverse transcriptase inhibitor (RTI)-containing therapy. Recombinant viruses containing the N-terminal part of RT in a wild type backbone were generated and genotypic and phenotypic resistance were analysed using the PhenoSense assay. RC was measured using four different approaches: viral infectivity, single cycle RC assay (PhenoSense RC), RT polymerase activity and hydroxyurea susceptibility. A large variation in baseline RC was observed. All failure recombinant viruses demonstrated genotypic and phenotypic resistance and generally conferred a decrease in RC when compared to their parental baseline virus. In conclusion, all approaches reported a reduction in RC for the majority of failure viruses, although the absolute effect differed for each assay. Comparison with a laboratory-adapted wild type virus instead of the parental baseline virus seems to result in an overestimation of the reduction in RC. Comparing RC results between baseline and failure samples from the same patient gives a better insight in the consequences of resistance on RC than when results are compared to a laboratory adapted reference virus. Introduction Multiple amino acid changes that decrease susceptibility to currently approved HIVdrugs have been described and are listed by the International AIDS Society, USA (IAS-USA) 1. Several studies have shown that these resistance-associated mutations will impact the viral replication capacity (RC), which might influence treatment efficacy 2. The impact of amino acid changes that confer resistance to the class of protease inhibitors on viral RC, in the absence of drugs, has been studied in great detail. Protease inhibitor resistance is usually characterized by the accumulation of resistance-mutations that are clustered in or near the substrate binding cleft of the enzyme. These primary amino acid changes cause a decrease in RC as a result of reduced proteolytic processing of Gag and Gag-Pol 3. During continuous (suboptimal) therapy, compensatory mutations can be selected. These compensatory changes, either located in protease or the protease cleavage sites, help the enzyme to adapt to the resistance-mutations in the substrate binding cleft or provide better peptide substrates for the mutant protease, respectively. In these ways, the compensatory changes can increase the RC of the PI-resistant virus

49 Replication capacity of RTI resistant HIV-1 In contrast to the situation with protease, much less is known about the effect of resistance-associated mutations in reverse transcriptase on RC. Most studies reported a reduction in RC for RTI resistant variants as well, although to a lower extent compared to PI resistant variants 3, Studies involving measurements of RC published so far use a great variety of assays to measure RC. The lack of a consensus approach on the in vitro determination of RC for HIV-1 isolates makes comparisons between studies difficult to interpret. Furthermore, RC is usually compared to a laboratory reference strain, such as HXB2, LAI or NL4-3. Given that several studies showed a wide distribution in RC of wild type (pre-therapy or baseline) viruses, it still remains to be determined whether it will be more appropriate to study the effect of amino acid changes in RT on RC by comparing the resistant virus with the patients own baseline (pretherapy) virus 18,19, In the present study we used paired samples from fifteen patients, one taken before and the other after extensive exposure to non-suppressive RTI-containing therapy. The reverse transcriptase gene was sequenced and recombinant viruses containing the patient-derived N-terminal part of the RT gene in a reference strain background (HXB2) were generated to eliminate putative effects of amino acid changes outside the target sequence. The RC of these viruses was measured using four different approaches, namely viral infectivity determination (TCID 50 /ml), single cycle replication capacity assay (PhenoSense RC assay), RT polymerase activity and hydroxyurea susceptibility. 49

50 Chapter 3 Table 1. Genotypic and phenotypic analysis of the recombinant viruses. Genotypic and phenotypic analysis of the baseline and failure recombinant viruses (RT amino acid ) as measured by the PhenoSense assay (Monogram Biosciences). Shown are the resistance-associated mutations as listed by the IAS (Fall 2006, marked in bold), and amino acid changes not listed by the IAS that have been associated with RTI-treatment. Unusual amino acid changes at known resistance-associated positions are indicated in grey. Fold increases in IC50 for (almost) all RTIs that are currently used in the clinic are indicated. Between brackets are the cut off values for each inhibitor which indicates a (clinical or biological) significant increase in IC50. Fold increases above this cut off are indicated in bold. ABC, Abacavir; ddi, Didanosine; FTC, emtricitabine; 3TC, Lamivudine; d4t, Stavudine; TFV, Tenofovir; ZDV, Zidovudine; DLV, Delavirdine; EFV, Efavirenz; NVP, Nevirapine; ins, insertion around amino acid 69; ND, not determined. 35I and 83K are negatively associated with RTI resistance. fold increase in IC50 compared to a reference virus [cut off] ABC ddi FTC 3TC d4t TFV ZDV DLV EFV NVP virus amino acid change compared to consensus B [4,5] [1,3] [3,5] [3,5] [1,7] [1,4] [1,9] [6,2] [3] [4,5] 1 baseline V35I/V,V60I/V failure M41L,K43K/E, E44E/A,V60I/V,D67N,V118I,K122E,I135T,L210W,T215Y baseline V35T,V60I,R83K,T200A,T215D failure V35T,M41L,V60I,D67N,R83K,V179V/I,M184V,G190G/E,T200A,L210W,R211K,T215Y >max >max baseline S162A,T200I.R211K failure M41L,K43E,E44E/A/D,D67N,L74L/I,V118I,K122K/T/Q/P,I135V,S162A,T200I,Q207K/N,H208H/Y,L210W,R211K,T215Y baseline K122K/E,I135I/T,S162C,T200I/T,R211K ND ND ND ND ND ND ND ND ND ND failure K103N,M184V,R211K,L228L/R >max >max >max baseline K101R failure K101R,V118V/A,M184V,G196G/R >max >max baseline V35I,E44E/A,R83K,K122E,Q207E,R211K,L228L/F,L283I failure V35I/R,R83K,M184V,Q207E,R211K,L283I >max >max baseline V35L,R83K,G196E,T200A,R211A,P225P/S failure V35L,R83K,I135L,M184V,G196E,T200A,R211A >max >max baseline Y115Y/H,K122E,T200A,R211K/R,L283I failure I31I/L,V35I,K65R,K122E,V179V/D,Y181C,M184V,T200A,E203E/K,P236P/S,L283I >max >max >max 3.7 >max 14 baseline I135I/V,R211K,F214L,H221H/Y failure V60I/V,D67D/N,V75I/M/V,K101E/K,K103R/K,V108I/V,V118I/V,K122E/K,I135I/R,S162D/G/N/S,Y181C/Y,G190A/G, E203D/E,Q207E/Q,H208H/Y,L210L/W,R211K,F214F/L,T215N/S/T/Y,D218D/E,K219K/Q,H221H/Y,L228H/L >max 15 baseline 211K/R failure V35L,M41L,E44D,D67G,T69D,V75M,F77F/L,A98A/G,K103K/N,V118I,K122E,I135I/V,M184V,G190A/G,T200T/A, E203K,H208Y,L210W,R211K,F214F/S,T215Y,K219K/R,K223E,F227F/L,L228H/R >max >max baseline R83K,K122K/E,R211S failure M41L,L74V,R83K/R,K103N,V118I,K122E,I135T,M184V,G196E,T200T/A,L210W,T215Y >max >max baseline D113D/G.I135I/V,T200I/T,E203E/D,L210S/L,R211A,T215A/C/D/G/S/Y failure V35L,M41L,K43N,E44D,ins KKDSTSTG,K70R, K122P,G196E,E203D,Q207K,L210W,R211A,T215Y ND ND ND ND ND ND ND ND ND ND 18 baseline V60V/I,V75A/V,83K,V90I,I202V,R211K failure T39A,M41L,K43K/E,D67N,K70K/R,L74V,V90I,K101E,V108I,V118I,K122E,V179I,Y181C,M184V,G190A,I202V,E203K,L210W, R211K,T215Y/C,D218E,K219E,K223T,L228H >max >max >max >max 19 baseline K122K/E,R211K//R/E/G failure T39A,M41L,E44D,D67N,L74V,K101Q,V106M,V108I,V118I,K122E,I135I/L,S162D,Y181C,Q207E,L210W,R211K,T215Y,K219N >max >max >max 20 baseline V35V/A/I/T,I135V,S162C/Y,I202V,R211G failure V35T,D67G,ins T, K70R,K122P,T200A,Q207A,R211K,T215F,K219Q,L228H

51 Replication capacity of RTI resistant HIV-1 Results Recombinant viruses All HIV-1 variants included in this cross-sectional study were subtype B and harboured at least one resistance-associated mutation in reverse transcriptase at the therapy failure timepoint 1. The RTI-resistance profiles of these viruses are shown in Table 1. Some viruses harboured multiple NRTI and/or NNRTI-resistance associated mutations, others contained a multi-drug resistance-conferring insertion in the fingers domain of RT. Three failure viruses (8, 9 and 10) revealed the presence of a single M184V amino acid change. In addition to known resistancemutations (marked in bold), all baseline and failure viruses showed the presence of amino acid changes associated with RTI-resistance that are not listed by the IAS These amino acid changes are listed in Table 1 as well. Phenotypic resistance profiles All baseline and failure viruses were phenotypically analysed using the PhenoSense assay (Monogram Biosciences). In accordance with the genotypic profiles all failure samples revealed (high-level) resistance to several RTIs for which resistance-mutations were selected, whereas all baseline samples demonstrated full susceptibility to all antiretroviral drugs tested (Table 1). Figure 1. Relative RC of the reference viruses as determined by various approaches. The four reference viruses with respectively a methionine (M), valine (V), isoleucine (I) or threonine (T) at position 184 in RT were analysed using four different RC approaches (viral infectivity, PhenoSense RC assay, RT polymerase activity and hydroxyurea susceptibility). relative replication capacity(%) Infectivity PhenoSense RC RC approach RT activity HU susc. 184M 184V 184I 184T Shown are the relative values in each assay compared to 184M (wild type) that was set at 100%. The HU IC 50 of >1000 µm for the 184M variant was set at 1000 µm to enable comparisons. Replication capacity approaches Reference viruses A set of four reference viruses harbouring a wild type methionine (M), valine (V), isoleucine (I) or threonine (T) at position 184 in RT (with a known effect on RC 70 ) was analysed in four different assays that measure RC aspects (Figure 1). The four reference viruses were analysed for viral infectivity, single cycle replication (PhenoSense RC assay), reverse transcriptase polymerase activity and 51

52 Chapter 3 hydroxyurea (HU) susceptibility. The results showed the same order of ranking for the four viruses (M>V>I>T) in each of the viral infectivity, RT polymerase activity and HU susceptibility assays. In the PhenoSense RC assay, the 184T demonstrated a slightly better RC than the 184I. Patient-derived recombinant viruses To determine the effect on RC caused by amino acid changes in the N-terminal part of RT that are selected during (suboptimal) therapy both baseline (=pretherapy) and failure recombinant viruses of fifteen patients were analysed in the four assays (Figure 2, 3, 4 and 5). In general, the failure recombinant viruses have a lower RC compared to the patients own baseline recombinant virus (Figure 2A, 3A, 4A and 5A). The relative decrease in RC, when compared to baseline, varies between the different viruses and different assays (Figure 2B, 3B, 4B and 5B). As shown in Figure 2A, 3A, 4A and 5A, the RC of the baseline recombinant viruses varies enormously. For the viral infectivity analysis, PhenoSense RC assay and RT polymerase activity assay, the variation in the results for the baseline viruses compared to the reference strain were in the range of 6-100%, % and % respectively. Most baseline samples revealed a lower RC compared to our wild type reference virus, except when analysed in the RT polymerase activity assays which demonstrated that the majority of baseline samples harboured a higher RT polymerase activity than the wild type reference virus. Most RC-assays use the RC of a laboratory-adapted wild type virus, such as HXB2, NL4-3 or a standard patient-derived virus as reference. In part C of Figure 2-5 we have determined the effect on RC when comparing the failure recombinant viruses with the wild type reference virus instead of the patients-derived baseline recombinant virus. Again, in general all failure samples revealed a decrease in RC when compared to the reference virus, but to a higher extent than when compared to the patients own baseline recombinant virus. In the PhenoSense RC assay all failure viruses revealed a decrease in RC when compared to the reference virus, whereas recombinant virus from patient 8, 14, 16, 18 and 19 did not show a decrease in RC when compared to their own baseline virus. For the RT activity determination virus 7, 9 and 14 showed no decrease in RC when compared to their own baseline virus. In contrast, when compared to the reference virus, virus 7 and 14 revealed a decrease in RC, whereas virus 9 and 10 demonstrated an increase in RC. 52

53 Replication capacity of RTI resistant HIV-1 A TCID50/ml Viral infectivity baseline failure recombinant virus TCID50/ml virus baseline failure 8 3.5E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E+03 A RC PhenoSense RC baseline failure recombinant virus RC(%) virus baseline failure B B percentage Viral infectivity baseline failure recombinant virus virus % percentage PhenoSense RC baseline failure recombinant virus virus % C C percentage Viral infectivity reference failure recombinant virus virus % percentage PhenoSense RC reference failure recombinant virus virus % Figure 2. Viral infectivity determination. The viral infectivity was assessed by determining the TCID 50/ml in MT2 cells by the method of Reed and Muench 80. All baseline and failure samples from the same patient were generated and titrated on the same day using the same materials for better comparison. The codon 184-reference viruses M, V, I and T harboured a titer of 1.2x10E5, 2.3x10E4, 7.0x10E3 and 7.0x10E3 TCID 50/ml respectively. A) raw data, B) data as a percentage of the patients own baseline sample, C) data as a percentage of the reference virus (HXB2). Figure 3. PhenoSense RC determination. RC as determined by the PhenoSense RC assay (Monogram Biosciences). The codon 184- reference viruses M, V, I and T harboured a RC of 87, 75, 20 and 45 % relative to the median of wild type variants. Patient 7 and 17 could not be analysed in this assay. A) raw data, B) data as a percentage of the patients own baseline sample, C) data as a percentage of the reference virus (HXB2). 53

54 Chapter 3 A A RT activity controlled for p24 amount Reverse transcriptase activity baseline failure recombinant virus RT activity virus baseline failure IC50 (µm) Hydroxyurea susceptibility IC50 (µm) virus baseline failure 8 >1000 > > > > > > > > > > > baseline failure 18 > > recombinant virus B B percentage Reverse transcriptase activity baseline failure recombinant virus virus % percentage Hydroxyurea susceptibility baseline failure recombinant virus virus % C percentage Reverse transcriptase activity reference failure recombinant virus virus % C percentage Hydroxyurea susceptibility reference failure recombinant virus virus % Figure 4. Reverse transcriptase polymerase activity determination. Reverse transcriptase activity was determined as described before 82. The codon 184-reference viruses M, V, I and T harboured a reverse transcriptase activity of 1.81, 1.22, 0.95 and 0.42 respectively. A) raw data, B) data as a percentage of the patients own baseline sample, C) data as a percentage of the reference virus (HXB2). Figure 5. Hydroxyurea susceptibility determination. Hydroxyurea susceptibility was determined for all baseline and failure samples as described by Bouchonnet et al. 77. All baseline samples and patient 8 failure sample revealed an IC 50 value of >1000 µm hydroxyurea, which were set at 1000 µm to enable comparisons. Patient 1 and 4 could not be analysed in this assay due to technical reasons. The codon 184- reference viruses M, V, I and T harboured an IC 50 value of >1000, , and µm respectively. A) raw data, B) data as a percentage of the patients own baseline sample, C) data as a percentage of the reference virus (HXB2). 54

55 Replication capacity of RTI resistant HIV-1 Correlation between the different assays To determine the inter-assay correlation, scatter plots were generated and linear trend lines were made. R 2 values were determined: Hydroxyurea susceptibility versus viral infectivity: , Hydroxyurea susceptibility versus RT activity: , Hydroxyurea susceptibility versus PhenoSense RC assay: , viral infectivity versus RT activity: , viral infectivity versus PhenoSense RC assay: , RT activity versus Phenosense RC assay: Although all assays demonstrated in general the same trends towards the reduction in RC, the relatively low R 2 values indicate a low level of correlation between the different approaches used in this study. 55

56 Chapter 3 Discussion Studies determining the effect of resistance-associated mutations in the (N-terminal part) of reverse transcriptase have been based on the results from several largely distinct assays, different viral constructs and reference viruses. Therefore it is hard or almost impossible to compare results between studies. In the present study we have included fifteen viruses, for which we determined the effect of emerging RTI mutations on RC by comparison to the own baseline. To our knowledge, this is the first study determining the effect on RC using a set of fifteen different recombinant RT-viruses by comparison with the parental pre-therapy virus, using four different approaches. In general, amino acids selected during failing antiretroviral treatment in the N-terminal part of RT resulted in a decrease in RC. A large variation of wild type RC between subjects was observed and most baseline samples have a lower RC compared to the wild type reference virus. These results are in accordance with previous studies which demonstrated extensive variation in RC between antiretroviral naïve HIV-1 isolates both upon analysis of complete and upon analysis of recombinant (PR/RT) viruses 18,19,49-51,71. Some baseline recombinant viruses revealed a higher reverse transcriptase polymerase activity compared to the reference virus. This higher activity might be attributable to the fact that only the N-terminal part of RT was analysed instead of the complete RT gene and/or that HXB2 has a relatively low RT activity. Whether these factors explain our observations needs further investigation. Although none of the baseline recombinant viruses has revealed the presence of a resistanceassociated mutation that might affect viral RC, all baseline viruses harboured several polymorphisms compared to the consensus B reference virus. Recent studies have identified novel substitutions that are associated with RTI-resistance 52-69, 72. Almost all viruses included in the current study harboured one or more of these changes. Furthermore, HIV-1 is able to select mutations in order to adapt to the host and escape the immune system 73. It seems plausible that these and other unidentified amino acid changes have an effect on the RC of wild type viruses. To study the effect of mutations in the N-terminus of RT on RC selected during antiretroviral therapy these baseline polymorphisms should be taken into account. We have demonstrated that the reduction in RC of the resistant viruses is slightly overestimated when compared to a wild type, laboratory adapted, reference strain instead of comparing it to the patient s own baseline sequence. In this study, we used four different approaches to measure RC. Although all approaches reported a reduction in RC for the majority of failure viruses the absolute effect in each assay differed and the correlation between the different assays was pretty low. The lack of correlation might be attributed to the different 56

57 Replication capacity of RTI resistant HIV-1 principles of the assays. The PhenoSense RC assay measures relative RC using a single cycle assay. The infectivity of the virus stock was determined using an endpoint dilution (TCID 50 /ml). Hydroxyurea susceptibility determination acts as a surrogate assay for the measurement of RC. In the reverse transcriptase activity assay solely the RNA-dependent DNA polymerase activity of RT is measured, and not its DNA-dependent DNA polymerase and RNase H activities and other intricate steps (e.g. strand transfers) of the natural reverse transcription reaction. From this, it is clear that each assay focuses on different processes that may be involved in RC. This study detected a general reduction in RC of RT-mutant virus compared to the parental wild type virus. A few exceptions were noticed which seems to be the result of the relatively low baseline RC, but further research is warranted to analyse these differences. To determine more subtle differences in RC, competition experiments could be performed. Such experiments are labour-intensive, timeconsuming and difficult to standardize. Given the pronounced differences determined between baseline and failure samples with the assays presented in this paper it was considered not relevant to also include competition experiments. Furthermore, we cannot exclude that regions outside the target sequence (Nterminus RT) affect RC. Some studies reported compensation of RC by regions outside the target sequence when a complete virus isolate was analysed compared to a recombinant virus 74,19. In this study we have assessed the impact of mutations in the N-terminal part of reverse transcriptase, the region that harbours all IAS- USA-listed resistance-associated mutations 1. Given the relatively small number of patients included in this study, we are unable to draw conclusions on the effect of specific RTI-resistance patterns on RC. An interesting subset of viruses in this study selected a single M184V mutation at failure compared to baseline (virus 8, 9 and 10). In most assays these viruses demonstrated the lowest change in RC when compared to viruses harbouring multiple RTI-resistance mutations. Especially for the virus derived from patient 8 hardly any effect on RC was observed in all assays that were performed. This was confirmed by our set of 184 mutant-reference viruses. The relative reduction in RC for failure virus 9 and 10 compared to their own baseline was lower than the decrease in RC of the 184V reference virus. Until now, no compensatory mutations have been described for the M184V change, although the negative effect of M184V has been demonstrated in various studies 75. Further research is warranted to determine if these viruses harbour compensatory changes that are selected de novo or were already present at baseline. For the other viruses in this study it would be interesting to investigate longitudinal samples to unravel the role of individual, emerging mutations with 57

58 Chapter 3 regard to resistance and RC as well to determine if compensatory changes are selected during treatment. Although the failure samples reveal a decrease in RC compared to baseline it is possible that the reduction in RC was even further decreased before the selection of compensatory changes 37,42. In conclusion, we have shown that different RTI-resistance profiles confer a decrease in RC that can be determined by various assays, although the correlation between results generated by the various approaches is low. The reduction in RC seems slightly overestimated when comparing with a reference wild type virus instead of the patients own baseline recombinant virus. Comparing RC results between baseline and failure samples from the same patient gives a better insight in the changes in RC than when compared to a laboratory adapted reference virus. Materials and Methods Viruses Fifteen paired samples of HIV-1 subtype B were selected that harboured (a) resistance-associated mutation(s) in the reverse transcriptase gene. From all viruses a pretherapy ( baseline ) sample and one sample during failing treatment ( failure ) were included. Generation of recombinant virus RNA was isolated from patient s plasma using the Qiagen kit according to the manufacturer s instructions and the reverse transcriptase (RT) encoding region was amplified using RT-PCR and nested PCR as previously described 76. Recombinant viruses that were used in the hydroxyurea sensitivity assays were generated as previously described 77. The amplified N-terminal part of reverse transcriptase was used to generate a recombinant virus stock containing amino acid 25 to 314 from RT in a wild type (HXB2) backbone representative for the virus population in the patient as described previously 78. Briefly, the amplified N-terminal part of RT was digested with 1 Unit Mlu NI (Roche) and 1 Unit Van91 I (Roche) and subsequently ligated O/N at 4ºC with a digested HXB2-vector lacking the N-terminus RT (amino acid ) using T4 ligase (Promega). To prevent religation of the vector the ligation product was digested with 1 Unit Asp I (Roche) before transformation into E.Coli JM109 High Efficiency Competent Cells (Promega) using a heat-shock at 42ºC. The transformed cells were cultured in Luria-Bertani (LB)- medium at 37ºC supplemented with 40 µg/ml ampicillin (Clamoxyl, GlaxoSmith Kline BV). The plasmid was isolated using the QIAGEN Plasmid Mini Kit (Qiagen) and 10 µg plasmid was used for transfection in 293T cells. Transfection was performed in T-25 culture flask with 90-95% confluent cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer s instructions. After 2 days, the virus was harvested by centrifugation for 10 minutes at 3000 rpm. These recombinant viruses were used in the TCID 50 analyses, RT activity assays and the corresponding plasmid was used for analysis in the PhenoSense RC assay. A set of four reference viruses encoding respectively a methionine (wild type), isoleucine, valine or threonine at position 184 (184M, 184I, 184V and 184T) in a wild type (HXB2) background was used to generate recombinant reference viruses

59 Replication capacity of RTI resistant HIV-1 Genotypic and phenotypic analysis All recombinant viruses were genotypically analysed and drug susceptibility for several reverse transcriptase inhibitors was determined using a recombinant virus approach as described previously (PhenoSense assay, 79 ). Replication capacity approaches Four assays were used to determine the RC of the recombinant viruses. Viral infectivity The amount of infectious virus in a viral stock was assessed by end-point dilution determination (each dilution in quadruplicate) in MT2 cells and determining the 50% tissue culture infectious dose per ml (TCID 50/ml) using the method described by Reed et al. 80. PhenoSense RC assay RC of all recombinant viruses was determined using a modification of the PhenoSense drug susceptibility assay (Monogram Biosciences) as described previously 51,81. In this single replication cycle assay, the luciferase activity expressed in infected cells in the absence of drugs is measured. RC is expressed relative to the median value calculated for a set of wild-type reference viruses (=100%). Reverse transcriptase polymerase activity Reverse transcriptase activity was determined for all baseline and failure viruses, essentially as described before 82. Briefly, RT was released from the virions by NP-40 treatment (final concentration 0.5%). The inactivated virus was incubated with 60 mm Tris-HCl (ph 7.8), 75 mm KCl, 5 mm MgCl 2, 1 mm EDTA, 0.1 % NP40, 5 µg/ml polya (poly[ra] 7000 (Boehringer (Roche)), 0.16 µg/ml oligo-dt (dt 15, Boehringer (Roche)), 8 µl 0.5 M DTT and 1-10 µl [α- 32 P]-TTP. Reactions were incubated at 37 C for two hours and subsequently spotted onto DEAE ion-exchange paper (DE81 paper, Whatman). The filter was washed three times with 5% Na2HPO4, two times with 96% ethanol, airdried and quantitated on a PhosphorImager. The RT activity is shown as a value relative to a control virus that was arbitrarily set at 1 and corrected for the amount of p24, as a surrogate for the amount of virions, that was present in the virus stock. Hydroxyurea susceptibility determination As surrogate for the measurement of the RC of HIV RT the susceptibility of recombinant viruses to hydroxyurea (HU) was assessed. HU results in a reduction of the intracellular deoxynucleotide concentration (especially datp) 83. It is hypothesized that viruses with a reduced RC will express an increased susceptibility to HU. The IC 50 for hydroxyurea was determined as described previously 77. Correlation between different approaches Scatterplots for each combination of replication capacity approaches were generated in Microsoft Excel 2002 using the raw data. A linear trend-line was generated and the R 2 value was calculated. Acknowledgements We would like to thank Prof. dr. I.M. Hoepelman and his staff for providing us the clinical samples. This study is financially supported by: EU-grant QLK2-CT (VIRULENCE) and Grant Number: 5 R44 AI (Monogram Biosciences) 59

60 Chapter 3 References 1. Johnson, V.A. et al. Update of the drug resistance mutations in HIV-1: Fall Top.HIV.Med. 14, (2006) 2. De Luca, A. et al. Association of HIV-1 Replication Capacity With Treatment Outcomes in Patients With Virologic Treatment Failure. J Acquir.Immune Defic.Syndr.45, (2007) 3. Quinones-Mateu, M.E. & Arts, E.J. HIV-1 fitness: implications for drug resistance, disease progression, and global epidemic evolution. HIV Sequence Compendium, (2001) 4. Mammano, F. et al. Resistance-associated loss of viral fitness in human immunodeficiency virus type 1: phenotypic analysis of protease and gag coevolution in protease inhibitor-treated patients. J Virol. 72, (1998) 5. Croteau, G. et al. Impaired fitness of human immunodeficiency virus type 1 variants with highlevel resistance to protease inhibitors. J Virol. 71, (1997) 6. Coffin, J.M. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267, (1995) 7. Kosalaraksa, P. et al. Comparative fitness of multi-dideoxynucleoside-resistant human immunodeficiency virus type 1 (HIV-1) in an In vitro competitive HIV-1 replication assay. J Virol 73, (1999) 8. Jeeninga, R.E. et al. Evolution of AZT resistance in HIV-1: the intermediate that is not observed in vivo has a replication defect. Virology 283, (2001) 9. Archer, R.H. et al. Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture. J Virol. 74, (2000) 10. Gerondelis, P. et al. The P236L delavirdine-resistant human immunodeficiency virus type 1 mutant is replication defective and demonstrates alterations in both RNA 5'-end- and DNA 3'- end-directed RNase H activities. J Virol. 73, (1999) 11. Imamichi, T. et al. High-level resistance to 3'-azido-3'-deoxythimidine due to a deletion in the reverse transcriptase gene of human immunodeficiency virus type 1. J.Virol. 74, (2000) 12. Deval, J. et al. Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations. J.Biol.Chem. 279, (2004) 13. Diallo, K. et al. Diminished RNA Primer Usage Associated with the L74V and M184V Mutations in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Provides a Possible Mechanism for Diminished Viral Replication Capacity. J Virol. 77, (2003) 14. Weber, J. et al. A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy. J Gen.Virol. 84, (2003) 15. Huang, W. et al. Amino acid substitutions at position 190 of human immunodeficiency virus type 1 reverse transcriptase increase susceptibility to delavirdine and impair virus replication. J.Virol. 77, (2003) 16. Maeda, Y. et al. Altered drug sensitivity, fitness, and evolution of human immunodeficiency virus type 1 with pol gene mutations conferring multi-dideoxynucleoside resistance. J Infect.Dis. 177, (1998) 17. Eggink, D. et al. Insertions in the beta3-beta4 loop of reverse transcriptase of human immunodeficiency virus type 1 and their mechanism of action, influence on drug susceptibility and viral replication capacity. Antiviral Res. 75, (2007) 18. Nicastri, E. et al. Replication capacity, biological phenotype, and drug resistance of HIV strains isolated from patients failing antiretroviral therapy. J Med.Virol 69, 1-6 (2003) 19. Bleiber, G. et al. Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1. J Virol 75, (2001) 20. Villena, C. et al. Relative fitness and replication capacity of a multinucleoside analogue-resistant clinical human immunodeficiency virus type 1 isolate with a deletion of codon 69 in the reverse transcriptase coding region. J Virol. 81, (2007) 21. Clark, S.A. et al. Reverse transcriptase mutations 118I, 208Y, and 215Y cause HIV-1 hypersusceptibility to non-nucleoside reverse transcriptase inhibitors. AIDS. 20, (2006) 60

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62 Chapter Fan, N. et al. A drug resistance mutation in the inhibitor binding pocket of human immunodeficiency virus type 1 reverse transcriptase impairs DNA synthesis and RNA degradation. Biochemistry. 35, (1996) 46. Hu, Z. et al. Virologic Characterization of HIV Type 1 With a Codon 70 Deletion in Reverse Transcriptase. J Acquir.Immune.Defic.Syndr. in press (2007) 47. Cong, M.E. et al. The fitness cost of mutations associated with human immunodeficiency virus type 1 drug resistance is modulated by mutational interactions. J Virol. 81, (2007) 48. Koval, C.E. et al. Relative replication fitness of efavirenz-resistant mutants of HIV-1: correlation with frequency during clinical therapy and evidence of compensation for the reduced fitness of K103N + L100I by the nucleoside resistance mutation L74V. Virology. 353, (2006) 49. Hellmann, N. et al. Modelling the effect of HIV replication capacity on treatment outcomes. Antiv. Ther. 7, S53 (2002) 50. Wrin, T. et al. Natural variation of replication capacity measurements in drug-naive/susceptible HIV-1. Antiv. Ther.apy 6, S20 (2001) 51. Campbell, T.B. et al. Relationship between in vitro human immunodeficiency virus type 1 replication rate and virus load in plasma. J.Virol. 77, (2003) 52. Ceccherini-Silberstein, F. et al. High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations. J.Virol. 79, (2005) 53. Vingerhoets, J. et al. TMC125 displays a high genetic barrier to the development of resistance: evidence from in vitro selection experiments. J Virol.79, (2005) 54. Brown, A.J. et al. Reduced susceptibility of human immunodeficiency virus type 1 (HIV-1) from patients with primary HIV infection to nonnucleoside reverse transcriptase inhibitors is associated with variation at novel amino acid sites. J Virol. 74, (2000) 55. Sarmati, L. et al. Failure of stavudine-lamivudine combination therapy in antiretroviral-naive patients with AZT-like HIV-1 resistance mutations. J Med.Virol. 65, (2001) 56. Montes, B. & Segondy, M. Prevalence of the mutational pattern E44D/A and/or V118I in the reverse transcriptase (RT) gene of HIV-1 in relation to treatment with nucleoside analogue RT inhibitors. J Med.Virol. 66, (2002) 57. Girouard, M. et al. Mutations E44D and V118I in the reverse transcriptase of HIV-1 play distinct mechanistic roles in dual resistance to AZT and 3TC. J Biol.Chem. 278, (2003) 58. Hertogs, K. et al. A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. Antimicrob.Agents Chemother. 44, (2000) 59. Yahi, N. et al. Mutation patterns of the reverse transcriptase and protease genes in human immunodeficiency virus type 1-infected patients undergoing combination therapy: survey of 787 sequences. J.Clin.Microbiol. 37, (1999) 60. Hanna, G.J. et al. Patterns of resistance mutations selected by treatment of human immunodeficiency virus type 1 infection with zidovudine, didanosine, and nevirapine. J Infect.Dis. 181, (2000) 61. De Luca, A. et al. Polymorphisms in the viral reverse transcriptase predict the evolution towards distinct thymidine analogue mutational patterns: a longitudinal analysis. Antiviral Therapy 11, S157-(2006) 62. Sturmer, M. et al. Correlation of phenotypic zidovudine resistance with mutational patterns in the reverse transcriptase of human immunodeficiency virus type 1: interpretation of established mutations and characterization of new polymorphisms at codons 208, 211, and 214. Antimicrob.Agents Chemother. 47, (2003) 63. Marcelin, A.G. et al. Impact of HIV-1 reverse transcriptase polymorphism at codons 211 and 228 on virological response to didanosine. Antivir.Ther. 11, (2006) 64. Stoeckli, T.C. et al. Phenotypic and genotypic analysis of biologically cloned human immunodeficiency virus type 1 isolates from patients treated with zidovudine and lamivudine. Antimicrob.Agents Chemother. 46, (2002) 65. Lu, J. et al. Effect of the Q207D mutation in HIV type 1 reverse transcriptase on zidovudine susceptibility and replicative fitness. J Acquir.Immune.Defic.Syndr.2005.Sep.1;40.(1): , (2005) 66. Saracino, A. et al. Impact of unreported HIV-1 reverse transcriptase mutations on phenotypic resistance to nucleoside and non-nucleoside inhibitors. J Med.Virol. 78, 9-17 (2006) 67. Svicher, V. et al. Involvement of novel human immunodeficiency virus type 1 reverse transcriptase mutations in the regulation of resistance to nucleoside inhibitors. J.Virol. 80, (2006) 62

63 Replication capacity of RTI resistant HIV Rhee, S.Y. et al. HIV-1 Protease and Reverse-Transcriptase Mutations: Correlations with Antiretroviral Therapy in Subtype B Isolates and Implications for Drug-Resistance Surveillance. J Infect.Dis. 192, (2005) 69. Precious, H.M. et al. Multiple sites in HIV-1 reverse transcriptase associated with virological response to combination therapy. AIDS 2000.Jan.7.;14(1): , (2000) 70. Keulen, W. et al. Initial appearance of the 184Ile variant in lamivudine-treated patients is caused by the mutational bias of human immunodeficiency virus type 1 reverse transcriptase. J.Virol. 71, (1997) 71. Bates, M. et al. Mutations in p6 Gag Associated with Alterations in Replication Capacity in Drug Sensitive HIV-1 Are Implicated in the Budding Process Mediated by TSG101 and AIP1. CROIabstract 121-(2004) 72. Berkhout, B. et al. Identification of alternative amino acid substitutions in drug-resistant variants of the HIV-1 reverse transcriptase. AIDS 20, (2006) 73. Goulder, P.J. et al. Evolution and transmission of stable CTL escape mutations in HIV infection. Nature.2001.Jul. 412, (2001) 74. Simon, V. et al. Infectivity and replication capacity of drug-resistant human immunodeficiency virus type 1 variants isolated during primary infection. J.Virol.2003.Jul.;77.(14): , (2003) 75. Perno, C.F. et al. Novel drug resistance mutations in HIV: recognition and clinical relevance. AIDS Rev. 8, (2006) 76. Masquelier, B. et al. Genotypic and phenotypic resistance patterns of human immunodeficiency virus type 1 variants with insertions or deletions in the reverse transcriptase (RT): multicenter study of patients treated with RT inhibitors. Antimicrob.Agents Chemother. 45, (2001) 77. Bouchonnet, F. et al. Quantification of the effects on viral DNA synthesis of reverse transcriptase mutations conferring human immunodeficiency virus type 1 resistance to nucleoside analogues. J Virol. 79, (2005) 78. van Maarseveen, N.M. et al. A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants. J Virol.Methods 133, (2006) 79. Petropoulos, C.J. et al. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob.Agents Chemother. 44, (2000) 80. Reed, L.J. & Muench, H. A simple method of estimating fifty percent endpoints. Am.J.Hyg 27, (1938) 81. Barbour, J.D. et al. Evolution of phenotypic drug susceptibility and viral replication capacity during long-term virologic failure of protease inhibitor therapy in human immunodeficiency virusinfected adults. J Virol. 76, (2002) 82. Back, N.K. et al. Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. EMBO J. 15, (1996) 83. Back, N.K. & Berkhout, B. Limiting deoxynucleoside triphosphate concentrations emphasize the processivity defect of lamivudine-resistant variants of human immunodeficiency virus type 1 reverse transcriptase. Antimicrob.Agents Chemother. 41, (1997) 63

64 Chapter 3 64

65 Chapter 4 Evolution of a novel 5-amino-acid insertion in the β3 β4 loop of HIV-1 reverse transcriptase M.C.D.G. Huigen 1, L. de Graaf 1, D. Eggink 1, R. Schuurman 1, V. Müller 2, A. Stamp 3, D.K. Stammers 3, C.A.B. Boucher 1 and M. Nijhuis 11 Published in: Virology 364, (2007) doi: /j.virol Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, the Netherlands 2 Institute of Biology, Eötvös Loránd University, Budapest, Hungary 3 Division of Structural Biology, University of Oxford, The Wellcome Trust Centre for Human Genetics, Oxford, UK

66 Chapter 4 Abstract HIV-1 isolates harbouring an insertion in the β3 β4 loop of reverse transcriptase (RT) confer high-level resistance to nucleoside analogues. We have identified a novel 5-amino-acid insertion (KGSNR amino acids 66 70) in a patient on prolonged nucleoside combination therapy (didanosine and stavudine) and investigated which factors were responsible for its outgrowth. Remarkably, only small fold increases in drug resistance to nucleoside analogues were observed compared to wild type. The insertion variant displayed a reduced replicative capacity in the absence of inhibitor, but had a slight replicative advantage in the presence of zidovudine, didanosine or stavudine, resulting in the selection and persistence of this insertion in vivo. Mathematical analyses of longitudinal samples indicated a 2% in vivo fitness advantage for the insertion variant compared to the initial viral population. The novel RT insertion variant conferring low levels of resistance was able to evolve towards a high-level resistant replication-competent variant. Introduction The human immunodeficiency virus type 1 (HIV-1) enzyme reverse transcriptase (RT) is an important target for antiretroviral treatment, due to its unique and essential role in the virus life cycle 1. Nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) compete with the natural dntps for incorporation in the growing proviral DNA chain 2. Once incorporated, further DNA synthesis is blocked, because NRTIs lack the 3 -hydroxyl group essential for chain elongation. Unfortunately HIV is able to develop resistance against all NRTIs. Crystallography studies have shown that the amino acid substitutions causing resistance are located around the nucleotide-binding site. They confer resistance against NRTIs via two mechanisms: discrimination between the nucleotide analogue and its natural counterpart (dntp) or enhanced excision of the incorporated nucleotide analogue 3,4. Continued treatment with NRTIs can lead to the development of cross-resistance to the complete drug class. Three pathways have been described that confer class resistance. Accumulation of mutations associated with NRTI cross-resistance, such as M41L, D67N, K70R, L210W, T215Y/F and K219Q/E, is the most prevalent pathway 5. Another pathway is referred to as the Q151M pathway, in which the Q151M substitution is selected, in combination with amino acid changes A62V, V75I, F77L and F116Y 6,7. The third pathway involves the selection of an insertion between codons 68 and 69 or 69 and 70 and has an estimated prevalence of less than 3% in therapy-failing patients These 66

67 Evolution of a novel 5-amino-acid insertion in HIV-1 RT insertions are mostly dipeptides, mainly SS, SG or SA, although single amino acid as well as three to eleven amino acid insertions have also been described Such insertions localize in the β3 β4 loop of the fingers subdomain of HIV-1 RT, which plays a key role in the functioning of the polymerase by positioning the template and incoming nucleotide 16. Genotypic analysis indicates that these insertions are usually duplications of prior codons, supporting the general opinion that generation of insertions is explained by slippage of RT at a pausing site 17. The selection of insertions usually occurs during treatment with at least one thymidine analogue (d4t and/or AZT). They generally appear in the background of specific thymidine associated mutations (TAMs), such as the M41L, K70R, L210W and T215Y/F changes and are highly associated with A62V, D67E/G and T69S substitutions 10,18,19. When accompanied by (a part of) these mutations the insertion complex invariably causes high-level resistance to all approved nucleoside analogues. The precise mechanism through which these insertions cause resistance is not clear. Although the insertion is able to increase the discrimination of a nucleotide analogue, the major mechanism of resistance for the insertion complex appears to be the increased excision of NRTIs from their terminated primer We have identified an HIV-1 variant harbouring a stable novel 5-amino-acid insertion in the fingers domain of RT in a patient failing on nucleoside combination therapy. In this study we have analyzed which factors drive the selection of this novel insertion variant. Results Genotypic analysis Longitudinal plasma samples of patient M17001 were genotypically analyzed as shown in Figure 1. At 263 weeks after start of highly active antiretroviral therapy (HAART) the viral population sequence (sample 5) revealed a very large, unique insertion of 15 nucleotides around codon 66 in RT. These 15 nucleotides encoded for amino acids KGSNR; an exact duplication of amino acids 66 to 70 (Figure 1B). This 5-amino-acid insertion was selected in the background of the D67G, K70R, V118I, K219Q and the unusual T69N and T215I amino acid changes. Several other mutations that are not associated with an insertion in the fingers domain of RT were present in the viral background as well (Figure 1). Selection of the insertion in RT did not coincide with substitutions in the C-terminal part of RT and/or protease (data not shown). 67

68 Chapter 4 A A 4B AZT 3TC RTV d4t ddi Saq/Inv HIV RNA (copies/ml) HIV RNA CD4 cells time (weeks) CD4 cells (10E3 cells/ml) amino acid in RT insertion wild type (consensus B) K T E K A V T V K K D S T K R K V K T K N D M G R F T K sample 1 K/R I N R K I L R H K L sample 2 K I S V K L sample 3 R I/V I G N R K Q I E L R K L Q sample 4 R A/G I/V I G N R K Q I E L R K L Q sample 4 clone 18 (S4C18) R G I I G N R K Q I E L R K L Q sample 5 R I I G N R KGSNR K Q I E L R K L I Q sample 5 clone 22 (S5C22) R A I I G N R KGSNR K Q I E L R K L I Q sample 6 R E R/T A/T I K/R G D N R KGSNR K Q I E L K/R K L F/T Q sample 6 clone 3 (S6C3) R A I G N R KGSNR K Q I E L R E K L Q sample 6 clone 4 (S6C4) R E R I G D N R KGSNR K Q I E L E K L F Q sample 6 clone 10 (S6C10) R G R I G D N R KGSNR K Q I E L R K L Q sample 7 R E R/T A/T I K/R G D N R KGSNR K/R Q I E L K/R L F/T Q B amino acid in RT consensus B K K K D S T K W R wild type AAG AAA AAA GAC AGT ACT AAA TGG AGA K K K G S N R W R sample 4 AAG AAA AAA GGC AGT AAT AGA TGG AGA K K K G S N R K G S N R W R sample 5 AAG AAA AAA GGC AGT AAT AGA AAA GGC AGT AAT AGA TGG AGA Figure 1. (A) Viral load, CD4+ count, treatment of patient M17001 and genotypic analyses. HIV-1 RNA copies and total CD4 counts were measured at several time points. The drug therapy is shown on top. At several time points during the treatment of patient M17001, as indicated by the black arrows, the plasma viral population was sequenced. For samples 4, 4A, 4B, 5 and 6 viral clones were generated and sequenced as well. (B) Nucleotide sequence of the 5-amino-acid insertion in RT. The nucleotide sequences and corresponding amino acids 64 to 72 of HIV-1 RT are shown for sample 4 and sample 5. The insertion is an exact duplication of the previous 15 nucleotides. AZT, zidovudine; 3TC, lamivudine; RTV, Ritonavir; d4t, stavudine; ddi, didanosine; Saq, Saquinavir. 68

69 Evolution of a novel 5-amino-acid insertion in HIV-1 RT During sustained treatment with stavudine (d4t) and didanosine (ddi) the T215I substitution evolved towards a mixture of phenylalanine and threonine, whereas the 5-amino-acid insertion remained stably present in the viral population (Figure 1). In this study we wanted to investigate which factor(s) is (are) driving the selection of this unique insertion, and therefore we determined its effect on replication capacity and drug susceptibility. Replication capacity of the novel insertion variant To investigate if the selection of the 5-amino-acid insertion variant is caused by an increase in replication capacity a recombinant virus clone was generated, which was representative of the viral population (sample 5 clone 22; S5C22 (insert)). For comparison a representative recombinant virus clone of sample 4 was generated (sample 4 clone 18; S4C18, Figure 1). Replication competition experiments demonstrated a negative effect on replication capacity, since S4C18 completely replaced the insertion-containing virus S5C22 (insert) clone (Figure 2). Moreover, in vitro evolution experiments demonstrated that the 5-amino-acid insertion was deleted after 65 days when the virus clone S5C22 (insert) was cultured in PBMC in the absence of RT inhibitors, while all other amino acid changes remained stably present (data not shown). Thus, in the absence of drug pressure the replication capacity of the insertion variant is decreased compared to its predecessor, indicating that another factor is driving the selection of this novel insertion. A % of virus in the culture time (days) B % of virus in the culture time (days) S4C18 S5C22 (insert) S4C18 S5C22 (insert) Figure 2. Competition experiments between S4C18 and S5C22 (insert). S4C18 (sample 4 clone, without insertion) was competed with S5C22 (sample 5 clone, with insertion) in PBMC for 5 passages. At several time points the percentage of both viruses in the culture was determined by sequencing as described in the Materials and methods section. Shown are two independent experiments that were started at a 50:50 (A) or 75:25 ratio (B) based on TCID 50. The variability is indicated by error bars representing the standard error of the mean (SEM). 69

70 Chapter 4 Drug susceptibility analysis To determine the impact of this novel insert variant on drug susceptibility the recombinant virus clones S5C22 (insert) and S4C18 were phenotypically analyzed. Results of at least duplicate experiments are shown in Table 1B. To our surprise, no high-level resistance for S5C22 (insert) could be determined for the three NRTIs tested. The moderate reductions in susceptibility to AZT, d4t and ddi were respectively 9-, 3-, and 2-fold. Our results were confirmed by the phenotypic assays from Monogram Biosciences (formerly ViroLogic; PhenoSense assay) and VIRalliance (PhenoScript assay). In these assays no high-level resistance could be observed for S5C22 (insert) as well (data not shown). Table 1. Drug resistance analyses for several viral HIV-1 clones derived from patient M A amino acid in RT consensus B K D S T K V T K S4C18 G N R I Q S5C22 G N R K G S N R I I Q S5C22-R70A G N R K G S N A I I Q S6C3 G N R K G S N R I Q S6C4 G D N R K G S N R I F Q S6C10 G D N R K G S N R I Q B PBMC AZT ddi d4t fold fold fold IC50 (nm) increase IC50 (nm) increase IC50 (nm) increase (mean ± SEM) in IC50 (mean ± SEM) in IC50 (mean ± SEM) in IC50 HXB2 (wt) 23 ± ± ± 73 S4C18 70 ± ± ± S5C ± ± ± 99 3 S6C3 288 ± ± ± S6C ± ± ± S6C ± ± ± C MT2 cells AZT fold IC50 (nm) increase (mean ± SEM) in IC50 S5C ± 807 S5C22-R70A 1179 ± Sample 4 clone 18 (S4C18) represents a viral clone without the insertion and sample 5 clone 22 (S5C22) contains the novel 5-aa insertion. Sample 6 clones correspond to the evolved virus variant in the patient. To determine the mechanism of resistance the second arginine at position 70 (of the insert) was replaced by a neutral alanine, resulting in virus clone S5C22-R70A. Shown are relevant amino acid changes (A) the mean IC 50 values compared to a wild type reference virus (HXB2) in PBMC (B) or compared to S5C22 in MT2 cells (C). AZT; zidovudine, d4t; stavudine, ddi; didanosine, wt; wild type 70

71 Evolution of a novel 5-amino-acid insertion in HIV-1 RT A small increase in resistance was observed for S5C22 (insert) compared to S4C18 for AZT in PBMC (9-fold compared to 3-fold), whereas no difference could be measured for d4t and ddi. However, small increases in drug resistance in PBMC were measured when we determined the viral replication capacity under drug pressure (Figure 3). The level of virus replication in the presence of serial dilutions of either d4t, ddi or AZT was assessed and for all drugs tested, an increased replication in the presence of inhibitor was observed for S5C22 (insert) as compared to S4C18. This means that at the higher drug concentrations the insertion variant (S5C22 (insert)) confers a higher replication capacity than the noninsertion variant (S4C18), indicating a small replicative advantage for the insertionvariant under d4t, ddi or AZT pressure in PBMC. Modelling of the 5-amino-acid insertion in RT and its putative mechanism of resistance A B HIV p24 (pg/100 µl) S4C18 S5C22 HIV p24 (pg/100 µl) S4C18 S5C22 10 no drug d4t concentration (nm) 10 no drug ddi concentration (nm) Figure 3. Replication capacity under drug pressure. The replication capacity of S4C18 and S5C22 was determined under a serial dilution of either d4t (A), ddi (B) or AZT(C). The amount of viral production in PBMC after 7 days, measured by P24 ELISA was plotted in a graph. Representative experiments are shown. In each experiment the virus production was measured in triplicate; the variability is indicated by error bars representing the standard error of the mean (SEM). C HIV p24 (pg/100 µl) S4C18 S5C no drug AZT concentration (nm) To obtain possible insights into the three-dimensional structure of this large insertion and its corresponding mechanism of resistance, a 3D model was generated (Figure 4). Because of the surface location of R70, the 5-residue insertion can be accommodated relatively easily in the RT structure. The minimized 71

72 Chapter 4 model gave indications of binding of the duplicated R70 to the triphosphate moiety of the substrate (dttp) and by extrapolation, to the inhibitor (AZT-TP) and also to the ATP used for excision of the blocked primer, in the binding mode modeled in previous reports 2,23. To determine if the second positively charged arginine is indeed responsible for the increase in resistance, a site-directed mutant was generated in which the arginine was replaced by an uncharged alanine (A) (S5C22-R70A; Table 1A). It was demonstrated that the replacement of the second arginine by an alanine indeed resulted in a two-fold decrease in IC 50 for AZT (Table 1C). A B Figure 4. Modelling of the 5-amino-acid insertion in reverse transcriptase. (A) Cartoon representation of the overall architecture of HIV-1 RT with bound template: primer DNA generated from 1RTD pdb coordinates. The p51 subunit is coloured light grey, the p66 subunit is dark grey, while the template:primer DNA strands are indicated by T and P respectively. The 5-amino-acid insertion (KGSNR) in the fingers domain of p66 is illustrated. The location of amino acids commonly associated with resistance to thymidine analogues (M41, T215 and W210) is indicated by arrows. (B) Cartoon illustration of the proposed interaction of residues within the 5-amino-acid insertion in the fingers domain of p66 generated from modelling studies. The main chains are shown in schematic form; the 5-amino-acid insertion is shown as a loop between K65 and R70; the template:primer strands are indicated by T and P respectively. The catalytic triad (D110, D185 and D186) along with residues K65 and R70 of the insertion and the dttp substrate are illustrated as ball and stick representations. A magnesium ion and the location of residues T215 and W210 are shown. Ionic and/or hydrogen-bonding interactions including that proposed for the duplicated R70 with the substrate triphosphate are shown as broken lines. In vivo fitness and evolution of the insertion variant To determine the fitness advantage of this novel insertion in vivo, viral clones were generated as described previously and genotypically analyzed to determine the percentage of virus in the population that contains the insertion. In sample 4A only 72