Antiviral Therapy 6:

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1 Antiviral Therapy 6: Increased drug susceptibility of HIV-1 reverse transcriptase mutants containing M184V and zidovudine-associated mutations: analysis of enzyme processivity, chain-terminator removal and viral replication Lisa K Naeger*, Nicolas A Margot and Michael D Miller Gilead Sciences, Foster City, Calif., USA *Corresponding author: Tel: ; Fax: ; lisa_naeger@gilead.com The presence of the HIV reverse transcriptase (RT) resistance mutation, M184V, induced by lamivudine and abacavir treatment results in increased tenofovir, adefovir and zidovudine susceptibility for HIV-1 with zidovudine-associated RT mutations in vitro. Treatment with oral prodrugs of tenofovir and adefovir has resulted in substantial HIV-1 RNA reductions in antiretroviralexperienced patient populations who have lamivudineand zidovudine-resistant HIV-1. An enzymatic analysis was undertaken to elucidate the mechanisms of altered drug susceptibilities of HIV-1 containing zidovudineassociated mutations in the presence or absence of M184V. The inhibition constants (K i ) for the active metabolites of tenofovir, adefovir and zidovudine did not vary significantly between recombinant mutant and wild-type RT enzymes. Although increased removal of chain-terminating inhibitors by pyrophosphorolysis and ATP-dependent unblocking correlated with reduced susceptibility of viruses with zidovudine-associated mutations, a reduction in the removal of chain-terminators was not observed, which would explain the increased drug susceptibility of mutants containing M184V plus zidovudine-associated mutations. However, analyses of single-cycle processivity of the mutant RT enzymes on heteropolymeric RNA templates showed that all M184Vcontaining mutant RT enzymes were less processive than wild-type RT, most notably for mutants expressing both zidovudine-associated mutations and M184V. Similarly, the in vitro replication capacity of a mutant virus expressing a zidovudine-associated mutation and M184V was significantly reduced compared with wild-type virus. The observed decrease in enzymatic processivity of the M184V-expressing RT enzymes might result in decreased viral replication, which then might contribute to the increased drug susceptibility of HIV-1 expressing these RT mutations. Introduction Combination antiretroviral therapy has resulted in significant reductions in HIV RNA for many HIVinfected patients. However, incomplete suppression of viral replication by antiretroviral drugs has also resulted in the development of resistant HIV and complex resistance profiles in patients. Interactions of multiple drug resistance mutations in reverse transcriptase (RT) can cause increased antiretroviral drug resistance and cross-resistance, but some drug resistance mutations can have favourable interaction effects. HIV containing foscarnet resistance mutations become hypersensitive to zidovudine and phenotypically reverse zidovudine resistance [1]. A more common example is the development of the lamivudine-associated M184V mutation that results in increased zidovudine susceptibility in vitro for zidovudine-resistant HIV containing the TY mutation in RT [2 4]. We have previously shown that the development of the M184V RT mutation in patient viruses also increases the in vitro susceptibility of HIV to two nucleotide analogue RT inhibitors, adefovir and tenofovir (PMPA) [5]. Tenofovir and adefovir are members of a new class of nucleotide-based antivirals that require only two phosphorylation steps by ubiquitous cellular enzymes to become active metabolites, adefovir diphosphate and tenofovir diphosphate [6,7]. This novel reduction in phosphorylation requirements allows drug activity in a wide variety of dividing and non-dividing cell types, including resting T cells and cells of the mono International Medical Press /01/$

2 LK Naeger et al. cyte/macrophage lineage [8,9]. Adefovir diphosphate and tenofovir diphosphate both act as competitive inhibitors of HIV-1 RT, and function as chain-terminators of viral DNA synthesis after incorporation into DNA [10]. The oral prodrug of tenofovir, tenofovir disoproxil fumarate (TDF), has shown antiviral activity in Phase I/II clinical trials against HIV [11 13] and is currently in Phase III clinical trials for HIV treatment. The oral prodrug of adefovir, adefovir dipivoxil, has demonstrated antiviral activity in Phase I/II/III clinical trials against HIV [14,15] and in Phase I/II clinical trials for the treatment of hepatitis B [16,17]. It is a more potent inhibitor of hepatitis B virus than of HIV, and is currently being developed for the treatment of hepatitis B at a dose of 10 mg once-daily. During the previous development of adefovir dipivoxil for the treatment of HIV, adefovir dipivoxil was added to patients antiviral regimen. Those with the combination of M184V and zidovudine-associated mutations in their HIV had improved plasma HIV RNA reductions compared with patients without M184V, which corresponded to the increased in vitro susceptibility of the patients HIV [18 20]. Altered drug susceptibility of RT mutants might be due to a number of possible mechanisms, including a change in binding affinity for the inhibitor or natural substrate, decreased fitness of the virus resulting from impaired enzyme function, or removal of chain-terminators by pyrophosphorolysis (the reverse reaction of nucleotide incorporation) and/or ATP-dependent primer unblocking [21,22]. In this study, we have investigated recombinant RT enzymes containing zidovudine-associated mutations D67N, K70R and TY to determine the mechanism of altered susceptibility to tenofovir, adefovir and zidovudine. We have examined inhibitor binding, removal of chain-terminating inhibitors by pyrophosphorolysis and ATP-dependent primer unblocking, and enzyme processivity. Additionally, we have investigated the M184V-mediated increased susceptibility to tenofovir, adefovir and zidovudine by analysing HIV-1 RT enzymes containing M184V with the zidovudine-associated RT mutations D67N, K70R and TY. Materials and methods Recombinant HIV production and antiviral susceptibility assays PCR fragments corresponding to the first 1000 bp of HIV RT and HIV RT mutants were amplified and cotransfected with the RT-deleted HIV-1 proviral molecular clone phxb RT (a gift from C Boucher) as previously described [5,23]. Replicationcompetent viruses generated by homologous recombination were harvested after 8 18 days when the cultures contained notable syncytia. The genotypes of the recombinant viruses were determined by RT PCR of viral supernatant followed by sequencing with an ALF Express automated DNA sequencer (Pharmacia, Piscataway, NJ, USA). Susceptibility of the recombinant mutant viruses and the wild-type HIV-1 molecular clone HXB2D to adefovir, tenofovir, zidovudine, lamivudine and ritonavir was assessed with a modified XTT-based assay in MT-2 cells as previously described [24]. All infections were done with cells at a multiplicity of infection (m.o.i.) of approximately 0.001, which resulted in equal levels of cell death in the absence of drug over the 5-day assay period. IC 50 values were calculated as an average from three to eight experiments. The RT mutant viruses all had the same susceptibility (0.02 µm) to the protease inhibitor ritonavir, since the protease genes are identical in the recombinant viruses. Recombinant RT construction, purification and kinetic analyses The wild-type RT expression construct prt66 [25] was a gift from M Wainberg. The pol sequences were amplified by PCR from the HXB2D molecular clone of HIV-1 and cloned into expression vector pkk223-3 (Pharmacia). RT mutants TY (), M184V (184), D67N/K70R (67/70), D67N/K70R/TY (), M184V/TY () and D67N/ K70R/M184V/TY () were generated by oligonucleotide-based site-directed mutagenesis and constructs were sequenced completely. The Escherichia coli strain JM109 was transformed with the wild-type or mutant constructs and induced with 1.5 mm isopropyl-β-d-thiogalactopyranoside. RT purification was done as described [26] using DEAE cellulose, phosphocellulose and poly (rc)-agarose column chromatography sequentially. The enzyme kinetic analyses were performed as described [27]. The reaction mixtures for the DNA-dependent DNA polymerase function contained 50 mm Tris-HCl (ph 7.8), 60 mm KCl, 10 mm MgCl 2, 5 mm DTT, 50 µm of each dntp, 150 µg/ml activated calf thymus DNA (Pharmacia) and Ci/mmol of the appropriate [ 3 H] dntp (Amersham, Arlington Heights, Ill., USA). Kinetic constants were determined by plotting [S] versus the initial rate data in a Michaelis Menten hyperbolic relationship curve and fitting to the equation f=v m x/(k m +x), fit f to y, using SigmaPlot Processivity assays Heteropolymeric HIV RNA template was prepared from a linearized phiv-pbs plasmid [28] using the Promega Ribo Maxi kit as described [29]. A second RNA template containing the truncated A/T rich International Medical Press

3 HIV-1 RT with M184V and zidovudine-associated mutations untranslated RNA4 region of the alfalfa mosaic virus together with the 5 -end of the coding sequence from human cytomegalovirus (HCMV) DNA polymerase was prepared as described [29,30]. Double stranded template/primer was prepared in batch by incubating the RNA templates (65 nm) with a DNA oligonucleotide primer (80 nm) for 10 min at 85 C, 10 min at 55 C, and then 10 min at 37 C. The sequence for the HIV-1 oligonucleotide primer (prpbs) was 5 -GTC CCT GTT CGG GCG CCA-3 and corresponded to the natural trna primer binding site. The HCMV pol oligonucleotide primer sequence was 5 -CCG CGA CCG CAC CGC CGG TCA-3. The processivity assays were carried out essentially as described [31]. Briefly, 10 nm of the wild-type or mutant RT was pre-incubated at room temperature for 10 min with 2 pmol of heteropolymeric template/primer (calculated in moles of primer) in a 10 nm DTT, 50 mm Tris-HCl (ph 6.8), 60 mm KCl, 1 mm EDTA and 10 mm MgCl 2 reaction buffer. The 50-µl reactions were started by adding the dntps with or without quenching [50 µg/ml poly(ra)/p(dt) (Pharmacia Biotech) or heparin (0.2mg/ml final)] at a final concentration of 50 µm dttp, dctp and dgtp; 50 nm [α- 33 P]dATP (500 Ci/mmol) and 450 nm cold datp. The concentration of the quenching poly(ra)/p(dt) was in 90-fold molar excess to the heteropolymeric template/primer. After 30 min at 37 C, the total incorporation of unquenched reactions was assessed in a filter-based assay where 15-µl aliquots were applied to Whatman 3-mm filters and washed three times for 10 min each in 5% tricholoroacetic acid, twice in 95% ethanol, then dried and counted in liquid scintillation fluid (Ready Safe; Beckman, Palo Alto, Calif., USA). The quenched reactions were stopped with 4X formamide loading buffer, heated for 2 min at 90 C, and electrophoresed in a 8 M urea-6% polyacrylamide gel. The processivity of the mutant and wild-type RT enzymes was determined by normalizing the amount of full-length product from quenched reactions obtained by phosphorimager analysis with total activity determined from the filter-based assay above. Two independent clones and protein preparations of RT and RT were evaluated. The efficiency of quenching, as determined by the addition of the quenching template/primer or heparin prior to the test template/primer, was greater than 99%. Chain-terminator removal assays A 16 nucleotide prpbs primer and 18 nucleotide prpbs primer described above were 5 end-labelled using [γ 32 P] ATP and T4 polynucleotide kinase. The labelled primers were annealed to phiv-pbs RNA transcript (1:1.2 ratio of primer to RNA) for 10 min at 85 C, 10min at 55 C, and 10 min at 37 C. The template/ 16-mer primer was incubated with wild-type RT heterodimer (920 µm) in RT buffer [50 mm Tris (ph 8.0), 60 mm KCl, 10 mm MgCl2], including 100µM dctp and 300 µm adefovir diphosphate or tenofovir diphosphate at 37 C for 30 min in order to produce an 18 nucleotide primer chain-terminated with adefovir or tenofovir, respectively. The template/18-mer primer was incubated with wild-type RT heterodimer in RT buffer, including 100 µm dctp and 250 µm zidovudine triphosphate at 37 C for 30 min to produce a 20 nucleotide primer chain-terminated with zidovudine. The end-labelled chain-terminated primers were purified on a 16% polyacrylamide/8 M urea sequencing gel, excised and eluted in 0.5 M ammonium acetate, 10 mm Tris (ph 7.5), 0.1 mm EDTA, 2 mm MgCl 2 and 0.01% SDS, followed by desalting through a NAP10 column (Pharmacia). The purified primers were then annealed to phiv-pbs RNA template as described above. Template/chain-terminated primer (20 nm) was incubated with nm wild-type or mutant recombinant RT enzymes (equivalent activity determined as described below) for 5 10 min at 37 C in RT buffer and then pyrophosphate (150 µm NaPPi final) or ATP (3.2 mm final) was added. Reactions were stopped by the addition of an equivalent amount of sequencing gel-loading buffer. Samples were heated for 5 min at 95 C, run on 16% polyacrylamide/8 M urea sequencing gel and analysed by phosphorimager. The proportion removed was determined by quantitating upper chain-terminated primer band divided by the total of all bands in the lane and subtracting from 100%. We determined that in our experiments, 15 min was within the linear range of the removal reaction of adefovir, tenofovir and zidovudine by both pyrophosphorolysis and ATP-dependent unblocking, and thus have made the chain-terminator removal comparisons for the different RT mutants at 15 min. No degradation of the primer band was detected when each RT enzyme was incubated with the template/primer for min at 37 C in the absence of ATP or NaPPi, thus ruling out exonuclease activity. Different enzyme:template/primer ratios (1:1 1:10) with wild-type RT were tested and no difference was seen between the different ratios with 10% of chainterminator removed in each case. The activity of the recombinant RT enzymes was determined by using the 5 end-labelled 16 nucleotide prpbs primer (80 nm) annealed, as described above, to phiv-pbs RNA (65nM) in RT buffer, incubating with wild-type or mutant RT for 5 min at 37 C and then adding 100 µm dctp, datp, dttp and ddgtp for 8 min at 37 C. Polymerization was terminated after incorporation of five nucleotides because of the incorporation of dideoxy Antiviral Therapy 6:2 117

4 LK Naeger et al. Table 1. Drug susceptibilities of reverse transcriptase mutants of HIV-1 IC 50 values (µm) Nucleoside-associated mutations in RT Adefovir Tenofovir Zidovudine Lamivudine HXB2D (wild-type) 10.5 (±1.7) 4.4 (±0.2) 0.10 (±0.02) 1.9 (±0.1) (±0.4) 4.0 (±0.2) 0.07 (±0.01) >100* 32.0 (±6.0)* 7.8 (±1.6) 0.70 (±0.13)* 2.1 (±0.1) 6.2 (±0.8) 2.3 (±0.3) 0.07 (±0.01) >100* 35.5 (±7.3)* 8.2 (±1.2) 5.10 (±1.40)* 7.4 (±0.9) 8.4 (±0.9) 4.3 (±0.5) 0.62 (±0.15) >100* IC 50 values from site-directed reverse transcriptase (RT) recombinant mutants are averages of three to eight experiments with the standard error. *Statistically significant change from HXB2D wild-type (P<0.05). Statistically significant change from or RT (P<0.05). GTP. Samples were electrophoresed on a 16% polyacrylamide/8m urea sequencing gel. Wild-type and mutant RT activity were equilibrated by measuring the amount of ddgtp-terminated product by the phosphorimager and adjusting for the extension of 10% of the primer to ddgtp-terminated product. This method of measuring a short extension product (5 nucleotides) avoided the influence of processivity defects in determining the activity of the RT mutants. Rescue of chain-terminated DNA synthesis assays were performed by incubating annealed RNA template/terminated primer (20 nm) and wild-type or mutant RT (17 nm) for 5 min at 37 C, adding 3.2 mm ATP, 100 µm dttp and ddgtp for the zidovudine-terminated primer, or 100 µm datp and ddctp for the adefovir and tenofovir-terminated primers at 37 C. Samples were loaded on a 16% acrylamide/8 M urea gel and the amount of rescued product was determined by phosphorimager analysis. HIV replication kinetics analyses Peripheral blood mononuclear cells (PBMC) from HIVnegative blood donors were isolated by density centrifugation, activated with 5 µg/ml phytohaemagglutinin-p (Sigma) for 2 days, frozen, thawed and then expanded in RPMI with 20% FBS (Irvine Scientific, Santa Ana, Calif., USA) and 20 U/ml recombinant human IL-2 (Boehringer Mannheim). After a total of 6days in culture, cells were infected with recombinant HIV at an m.o.i of Cells were washed three times with PBS 18 h later, and then recultured in IL-2 containing RPMI for 3 weeks. No additional PBMC were added to the cultures. Supernatant samples were removed every 2 3 days during media exchanges for 21 days and evaluated for p24 concentration by ELISA (Coulter-Immunotech, Westbrook, Me., USA). Results Drug susceptibility Antiviral drug susceptibility assays were performed on site-directed recombinant HIV-1 RT mutant viruses using adefovir, tenofovir, zidovudine and lamivudine. The TY () recombinant HIV-1 mutant was threefold less susceptible to adefovir and sevenfold less susceptible to zidovudine than HXB2D wild-type (Table 1). The D67N/K70R/TY () recombinant HIV-1 mutant was threefold less susceptible to adefovir but 50-fold less susceptible to zidovudine than the wild-type. Both the and HIV-1 mutants had a less than twofold decrease in susceptibility to tenofovir as compared to wild-type. The M184V (184) RT mutant demonstrated a slight increase in susceptibility to adefovir, tenofovir and zidovudine when compared to HXB2D wild-type (Table 1). The addition of M184V mutation to the mutant HIV-1 expressing the RT mutation resulted in wild-type susceptibility levels for tenofovir, adefovir and zidovudine. The addition of M184V to resulted in restoration of wild-type susceptibilities for tenofovir and adefovir, but sixfold resistance to zidovudine was retained. The drug susceptibility results with these recombinant RT mutant viruses are similar to the drug susceptibility results obtained from patient viruses containing these mutations in a background of additional RT mutations [5]. Relative binding affinity Binding of the chain-terminating inhibitors or the natural substrate (dntp) might be affected by mutations in RT. Therefore, we analysed inhibitor binding of the E. coli expressed HIV-1 RT mutant enzymes, 184,,, and by determining the inhibitor dissociation constant (K i ) for adefovir diphosphate, tenofovir diphosphate, lamivudine triphosphate and zidovudine triphosphate, the active metabolites for these RT inhibitors. The affinity of the RT enzyme for the natural dntp substrate was approximated by determining the Michaelis constant (K m ). The K i /K m ratio, a measure of the relative inhibitory capacity, was calculated for the corresponding dntp (datp for adefovir diphosphate and tenofovir diphosphate, dctp for lamivudine triphosphate, and dttp for zidovudine triphosphate). As expected, the E. coli expressed 184 mutant HIV- 1 RT had significantly reduced binding to lamivudine International Medical Press

5 HIV-1 RT with M184V and zidovudine-associated mutations Table 2. Relative binding affinities of recombinant wild-type and mutant HIV-1 reverse transcriptase enzymes Adefovir diphosphate Tenofovir diphosphate Zidovudine triphosphate Lamivudine triphosphate Recombinant HIV-1 RT K i * (µm) K i /K m K i (µm) K i /K m K i (µm) K i /K m K i (µm) K i /K m Wild-type ND ND 67/70 ND ND ND ND ND *K i values are averages of multiple experiments with an average standard error of 0.05 for adefovir diphosphate and tenofovir diphosphate, 0.03 for zidovudine triphosphate and 0.3 for lamivudine triphosphate. K m values for wild-type, 184,,, 67/70, and, were 0.31, 0.49, 0.48, 0.34, 0.43, 0.33 and 0.45 µm, respectively, for datp,and 0.49, 0.3, 0.52, 0.61, 0.61, 0.78 and 0.84 µm, respectively, for TTP. ND, not determined; RT, reverse transcriptase. triphosphate with a 12-fold increase in K i value and 19-fold increase in K i /K m ratio compared to wild-type RT (Table 2). However, the 184 RT mutant and wildtype RT had similar K i values using adefovir diphosphate, tenofovir diphosphate and zidovudine triphosphate (Table 2). The K i values for the wild-type RT and the and RT mutant enzymes for adefovir and tenofovir diphosphate were all equivalent, with similar K i /K m ratios. The K i values of the and RT mutants for zidovudine triphosphate were about twofold higher than the K i of wild-type RT, which is consistent with other reports published previously that show a slight decrease in zidovudine triphosphate binding [32 34], but the K i /K m ratios were similar to wild-type. Therefore, changes in relative binding affinities for the inhibitors do not seem sufficient to explain the observed tenofovir, adefovir and zidovudine susceptibility shifts for the HIV RT mutants with the zidovudine-associated Figure 1. Removal of chain-terminator inhibitors by pyrophosphorolysis (a) (b) (c) 184 Removal of adefovir diphosphate (a), tenofovir diphosphate (b), and zidovudine triphosphate (c) by reverse transcriptase (RT) mutants 184,,, and, and wild-type () RT evaluated at 0, 5, 15 and 30 min following addition of 150 µm NaPPi. mutations. The and recombinant RT mutant enzymes had equivalent K i /K m ratios to the and RT mutants and wild-type RT (Table 2). Results with virion extracted RT enzymes from and viruses and patient viruses that included additional zidovudine-associated mutations also exhibited no M184V-mediated change in Ki/Km ratios for adefovir diphosphate and zidovudine triphosphate (data not shown). Our results demonstrated that the RT enzymes with the M184V mutation have comparable binding affinities to the TY-containing RT mutants for adefovir diphosphate, tenofovir diphosphate and zidovudine triphosphate. Thus, a difference in relative binding affinity was not primarily responsible for the increase in adefovir, tenofovir and zidovudine susceptibility of the M184V-containing RT mutants. Removal of chain-terminators by pyrophosphrolysis and ATP-dependent unblocking Pyrophosphorolysis is the reverse reaction of nucleotide incorporation, where removal of chain-terminators can be mediated by HIV-1 RT using a pyrophosphate acceptor molecule. This reaction, as well as nucleotidedependent primer unblocking (a similar mechanism), which uses nucleoside triphosphates (such as ATP) as acceptor molecules, have been proposed to remove nucleoside chain-terminators and thus contribute to resistance [21, 22]. We have analysed the removal of the chain-terminators, adefovir, tenofovir, and zidovudine, by these two mechanisms. First, we examined the pyrophosphate-dependent mechanism (pyrophosphorolysis) using physiological concentrations of NaPPi (150 µm) (Figures 1 and 2). At 15 min, the RT mutant removed an average of 36% of zidovudine from zidovudine-terminated primers following addition of pyrophosphate, and the RT mutant was even more efficient, removing an average of 53% of the zidovudine from zidovudine-terminated primers. The and RT mutants also showed increased removal of tenofovir and adefovir compared to wild- Antiviral Therapy 6:2 119

6 LK Naeger et al. Figure 2. Quantitation of chain-terminator removal at 15 min following addition of 150 µm NaPPi (a) (b) (c) % Removed at 15 min % Removed at 15 min % Removed at 15 min Graphs show the average from at least three experiments with standard deviation of (a) adefovir diphosphate (b) tenofovir diphosphate and (c) zidovudine triphosphate removed at 15 min from terminated primers with reverse transcriptase mutants 184,,, and, and wild-type (). type RT, but resulted in less than 25% removal at 15 min, consistent with the minor changes in tenofovir and adefovir drug susceptibility for these HIV mutants (Figure 2 and Table 1). Overall, the pyrophosphate dependent removal of zidovudine by all the analysed RT enzymes was more efficient than the removal of either adefovir or tenofovir. When removal from multiple experiments was averaged at 15 min, wild-type RT had removed zidovudine triphosphate from almost 20% of zidovudine-terminated primers, but had removed 10% or less of adefovir and tenofovir diphosphate from adefovir- and tenofovir-terminated primers. When ATP was added as the acceptor for chainterminator removal, the removal of tenofovir or adefovir chain-terminators by wild-type RT or any of the RT mutants was not detectable (Figure 3). Because we found no detectable removal of tenofovir or adefovir when 3.2 mm ATP was added for incubation periods up to 90 min (data not shown), the ATP-dependent unblocking mechanism does not appear to remove tenofovir or adefovir effectively. In contrast, zidovudine was readily removed by wild-type RT and all the RT mutants within 15 min. The and RT mutants were able to remove zidovudine more efficiently, threeand sixfold, respectively, compared to wild-type RT (Figures 3 and 4). Together with the results from the pyrophosphorolysis experiments, these results suggest that zidovudine removal by the and RT mutants may contribute to the significant decrease in susceptibility of these RT mutants to zidovudine. We assessed whether the increase in adefovir, tenofovir, and zidovudine susceptibility of M184V -containing RT mutants might be explained by a decrease in pyrophosphorolysis or ATP-dependent unblocking. The 184 RT mutant showed a small decrease of less than twofold for removal of adefovir, tenofovir and zidovudine compared to wild-type RT (Figures 1 and 2). Figures 1 and 2 also show the pyrophosphorolysis activity of the and RT mutants compared to and for the removal of adefovir, tenofovir and zidovudine from terminated primers. Although the RT mutant showed an apparent decrease of 1.5-fold in removal of adefovir by pyrophosphorolysis compared with, the mutant did not show a similar decrease in the removal of tenofovir or zidovudine (Figures 1 and 2). Furthermore, the RT mutant consistently showed no decrease in the pyrophosphate-dependent removal of adefovir, tenofovir or zidovudine compared with the RT mutant (Figures 1 and 2). There was also no decrease in ATP-dependent unblocking of zidovudine-terminated primers by and RT mutant compared with the and RT mutants (Figures 3 and 4). Because there was no International Medical Press

7 HIV-1 RT with M184V and zidovudine-associated mutations Figure 3. Removal of chain-terminator inhibitors by ATPdependent unblocking 184 Figure 4. Quantitation of zidovudine removal from zidovudine-terminated primers at 15 min following addition of 3.2mM ATP 50 (a) 40 (b) (c) Percent removed Removal of adefovir diphosphate (a), tenofovir diphosphate (b) and zidovudine triphosphate (c) by reverse transcriptase (RT) mutants 184,,,, and, and wild-type () RT evaluated at 5, 15 and 30min for adefovir and tenofovir and 0, 15 and 30 min for zidovudine following addition of 3.2 mm ATP detectable removal of adefovir and tenofovir from their respective terminated primers by ATP (Figure 3), quantitation was not possible for these terminated primers. In a variation of the chain-terminator removal assay, the ability of the RT mutants to reinitiate or rescue chain-terminated DNA synthesis was determined by adding ATP to remove the chain-terminator and measuring the extension to the next template position. In these assays, the M184V-containing RT mutants did not show a decrease in rescue of zidovudine-terminated DNA synthesis compared to the and RT mutants (data not shown). Thus, a significant decrease in pyrophosphorolysis or ATP-dependent primer unblocking does not seem to explain the increased adefovir, tenofovir or zidovudine susceptibility of M184V-containing RT mutants. Processive DNA synthesis To further analyse the potential mechanism for altered drug susceptibility of the M184V-containing recombinant RT mutants, we measured the processivity of our recombinant RT enzymes. DNA synthesis by HIV RT proceeds with multiple pauses and repeated cycles of association and dissociation of the enzyme from the template/primer [35]. Therefore, single-cycle processivity of wild-type RT and mutant recombinant RT enzymes was assayed by adding an excess of a quenching template, or heparin, to a heteropolymeric RNA template/dna oligonucleotide primer. The amount of 191-bp full-length product produced from an HIV RNA template in quenched reactions (Figure 5a and 5b) normalized to total unquenched enzyme activity Graph shows zidovudine removal averaged from at least three experiments with standard deviation by ATP-dependent unblocking from zidovudineterminated primers with reverse transcriptase (RT) mutants 184,,,, and, and wild-type () RT. determined from filter-based assays, was used to monitor single-cycle processivity of each of the recombinant RT enzymes (Figure 6). Reactions that did not include quenching template or heparin showed that the RT mutants had equivalent polymerization activity (Figure 5c). The 184 and recombinant RT enzymes both produced slightly less full-length product than wild-type RT in quenched reactions (Figures 5a and 6a). Analysis of the 67/70 and mutant RT enzymes also demonstrated a slight reduction in amount of full-length 191-bp product produced (Figures 5b and 6a). However, the addition of the M184V mutation to and RT mutants resulted in a substantial 1 log 10 reduction of full-length product produced (Figures 5a, 5b and 6a). RT mutants containing the TF/Y mutation have previously been reported to have increased processivity [22,36], yet we have analysed two independent clones and protein preparations of the TY RT mutant in the processivity assay and both demonstrated a slight decrease in processivity. For comparison, two additional recombinant RT mutants, TF/K219Q and D67N/K70R/TF/ K219Q, showed increased processivity as previously reported (Figure 6b) [22]. These results suggest that the presence of the K219Q with the TF or TY may contribute to increased processivity. Our processivity results on an HIV-derived RNA template were also confirmed with a second heteropolymeric RNA Antiviral Therapy 6:2 121

8 LK Naeger et al. Figure 5. Analysis of single-cycle processivity of site-directed recombinant RT mutants Figure 6. Single-cycle processivity normalized to total activity (a) / / bp Percentage of wild-type RT TY M184V M184V/ TY D67N/ K70R D67N/ K70R/ TY D67N/ K70R/ M184V/ TY (b) Single-cycle processivity of each of the Escherichia coli purified recombinant reverse transcriptase (RT) enzymes (a) wild-type () RT (lane 1), 184 (lane 3), (lane 4), and (lane 5). (b) RT (lane 6), 67/70 (lane 7), (lane 8), and (lane 9) were assayed on a heteropolymeric HIV RNA template in the presence of excess quenching template. Two times the amount of RT used in lane 1 was also included as a control for template excess (lane 2). (c) Continuous polymerization for and RT mutants was analysed on a heteropolymeric HIV RNA template with no quenching. All samples were electrophoresed on an 8 M urea-6% polyacrylamide gel. Full-length product for the HIV RNA template is 191 nucleotides. template derived from HCMV, which allows for efficient procession to its full-length 98-bp product with fewer pauses (data not shown). Viral replication kinetics The replication capacity of the 184, and HIV recombinant mutant viruses was tested in activated primary PBMC cultures. The and 184 mutant viruses replicated similarly to the wild-type virus with only slightly diminished replication capacity (Figure 7). However, the double mutant virus was markedly delayed in its onset of viral production and never achieved the level of virus production observed Percentage of wild-type RT TF/ K219Q D67N/ K70R/ TF/ K219Q (a) Single-cycle processivity results of wild-type () reverse transcriptase (RT), and RT mutants M184V, TY, M184V/TY, D67N/K70R, D67N/K70R/TY and D67N/K70R/M184V/TY from multiple independent experiments were averaged by quantitating full-length product from phosphorimager analyses and normalizing to unquenched filter assay data (See Materials and methods). All values are expressed as a percentage of RT with the percent standard deviation. (b) The average of two independent experiments with the percent standard deviation of single-cycle processivity results of the recombinant RT mutants, TF/K219Q and D67N/K70R/TF/K219Q (obtained from M Parniak) compared with RT. with the other viruses (Figure 7). Thus, in these experiments, the M184V or TY mutation alone in RT had only a modest affect on viral replication capacity, but the combination of the M184V mutation with the TY mutation in HIV RT resulted in severely diminished viral replication capability. The virus that grew out International Medical Press

9 HIV-1 RT with M184V and zidovudine-associated mutations of the infection at day 20 was analysed by sequencing, and determined to have the M184V and TY mutations with no additional mutations. Discussion Antiviral drug susceptibilities of site-directed recombinant viruses expressing the M184V, TY and D67N/K70R RT mutations were determined, individually and together, for tenofovir, adefovir and zidovudine. The and recombinant viruses have a two- to threefold decrease in susceptibility to adefovir and tenofovir, but a seven- and 50-fold decrease, respectively, in susceptibility to zidovudine. The addition of the M184V mutation to the TY-containing viruses results in viruses that have wild-type susceptibility to these drugs except for the mutant virus, which still has a sixfold decrease in susceptibility to zidovudine. Enzymatic analyses of the and RT mutants were performed to determine the mechanism for the decreased susceptibility to these inhibitors. The RT mutants and wild-type RT had equivalent K i values and K i /K m ratios for the inhibitors, and their corresponding substrates, indicating that changes in inhibitor binding are not responsible for the changes in susceptibility to the three drugs analysed. This lack of correlation between relative K i and viral resistance is similar to results published for zidovudine resistance [32 34]. Recent work has suggested a new mechanism of how zidovudine resistance occurs with these mutations Figure 7. Kinetic analysis of virus production p24 Concentration (ng/ml) Days post infection m.o.i.=0.01 Replication kinetics of HIV reverse transcriptase (RT) mutants, 184, and wild-type RT were determined following infection at a multiplicity of infection (m.o.i.) of 0.01 on MT-2 cells by measuring the p24 concentration (ng/ml) by ELISA every 2 3 days for 21 days. These results are representative of five independent experiments. [21,22,37,38]. In this model, an increased rate of the chain-terminating zidovudine that results from pyrophosphorolysis (reverse nucleotide polymerization) or nucleotide-dependent unblocking (a similar mechanism), could account in part for zidovudine resistance with these mutations. We observed removal of adefovir, tenofovir and zidovudine chain-terminator inhibitors by pyrophosphorolysis with wild-type and mutant RT enzymes, but in each case removal of zidovudine was greater than that of either adefovir or tenofovir. Removal of the chain-terminator inhibitors by this mechanism increased for the and RT mutants by two- to threefold compared with wild-type RT. In similar excision experiments using ATP as an acceptor molecule, tenofovir and adefovir were not detectably removed by wild-type or mutant RT, whereas zidovudine was removed sixfold more efficiently at 15 min by the RT mutant compared with wild-type RT. Consistent with previous reports, removal of zidovudine from zidovudine-terminated primers by both pyrophosphorolysis and ATP-mediated unblocking was more efficient when the RT mutant enzyme contained the D67N and K70R mutations [22,37]. Interestingly, the D67N and K70R mutations did not have this effect on removal of adefovir and tenofovir from terminated primers. The most physiologically relevant acceptor molecule for removal of chain-terminators as a mechanism of resistance in vivo is currently a matter of discussion. It has been reported that physiological concentrations of pyrophosphate are below the K m for the reverse reaction [39,40]. However, the K m for ATP is between 0.7 and 4.3 mm, which is in the physiological range for ATP, suggesting that ATP is the most likely acceptor for the chain-terminator removal reaction in vivo [41, 42]. Both mechanisms can result in unblocked chain-termination and our results show that both nucleotides, adefovir and tenofovir, are less efficiently removed than zidovudine with either mechanism. These results are consistent with the drug susceptibility changes with these mutants showing strong zidovudine-resistance, threefold adefovir-resistance and less than twofold changes in susceptibility to tenofovir. We performed further analyses to explore the mechanisms for the observed M184V-mediated increase in susceptibility to tenofovir, adefovir, and zidovudine. Enzymatic analysis of and RT mutants demonstrated that their relative K i values for adefovir diphosphate, tenofovir diphosphate and zidovudine triphosphate did not differ significantly from and RT mutants. Therefore, changes in relative binding affinities do not explain the increase in susceptibility of the M184V-containing viruses to these drugs. A decrease in removal of the chain-terminating RT inhibitors might contribute to Antiviral Therapy 6:2 123

10 LK Naeger et al. the increased susceptibility of M184V-containing HIV RT mutants. Therefore, we have addressed whether the addition of the M184V mutation to RT with zidovudine-associated resistance mutations results in a decrease in chain-terminator removal through pyrophosphate and ATP-dependent unblocking. Götte et al. [43] previously reported that the M184V RT mutant in the absence of zidovudine mutations has decreased removal of zidovudine. We observed that the M184V mutant had a slight decrease (<2-fold) in removal by pyrophosphorolysis of adefovir, tenofovir, and zidovudine compared with wild-type RT. Although the RT mutant had a decrease in the removal of adefovir through pyrophosphorolysis compared with the RT mutant, the addition of M184V to did not result in decreased adefovir removal. Moreover, the and RT mutants did not show a decrease in the removal of tenofovir or zidovudine by pyrophosphorolysis compared to or. Finally, there was no decrease in the removal of zidovudine by the ATPdependent unblocking mechanism of the RT mutants by the addition of the M184V mutation. Thus, our results suggest that a reduction in the rate of chainterminator removal does not contribute significantly to the increased susceptibility to adefovir, tenofovir or zidovudine observed with the M184V mutation in the background of these zidovudine-associated mutations. However, we have found that the M184Vcontaining RT mutants do have a significant defect in processivity, which is strongest in the background of zidovudine mutations. Previously, others have shown that the M184I and M184V RT mutants in the absence of zidovudine resistance mutations are less processive than wild-type RT in vitro using homopolymeric templates [44]. We have extended that observation and demonstrated that RT mutants with M184V and zidovudine resistance mutations have reduced processivity with heteropolymeric RNA templates. We observed that all of our recombinant RT mutant enzymes demonstrated minor decreases in processivity compared to wild-type RT. However, the addition of the M184V mutation to and resulted in RT enzymes clearly more defective in processivity than their parent RT enzymes. In fact, the individual defects in processivity of the 184 and RT mutants seemed additive in the double mutant. Back et al. [44] showed that the decrease in processivity of the M184I and M184V RT mutants in the absence of zidovudine-resistance mutations correlates with decreased viral replication of the lamivudine-resistant viruses in certain in vitro conditions. We have demonstrated that RT enzymes with both M184V and zidovudine resistance mutations are less processive than wild-type RT, and that the viral replication of the recombinant mutant virus is appreciably reduced compared with wild-type virus and the single RT mutants. Additionally, we have previously shown a consistent decrease in replication kinetics of eight patient-derived recombinant HIV pairs from patients developing the M184V mutation in a variety of nucleoside-resistant backgrounds [5]. M184V mutant viruses also have decreased fitness based on clinical data, which demonstrate that patients in monotherapy trials do not seem to return completely to baseline HIV RNA levels when they acquire the M184V mutation in their HIV and, after removal of lamivudine therapy, the HIV reverts to wild-type over time [44, 45]. Thus, conferring decreased fitness to HIV by maintaining the M184V mutation may contribute to lower viral RNA levels and increase the clinical benefit of antiviral drugs that have increased efficacy towards these mutant viruses. In conclusion, our results indicate that changes in relative binding affinities to RT inhibitors and the removal of these RT inhibitor chain-terminators by pyrophosphorolysis and ATP-dependent unblocking do not play a major part in M184V-mediated increased susceptibility to adefovir, tenofovir or zidovudine. However, the M184V-containing RT mutants are substantially less processive than wild-type RT and TY-containing RT mutants. This decrease in processivity may contribute to the significant reduction in viral replication capacity of the RT mutant and might have a yet unidentified role in the increase in tenofovir, adefovir and zidovudine susceptibility. We have confirmed that pyrophosphorolysis and ATP-dependent unblocking are potential mechanisms of zidovudine resistance. Additionally, we have shown an increase in removal of zidovudine compared with tenofovir and adefovir by pyrophosphorolysis and ATP-dependent unblocking which is in agreement with the susceptibility shifts seen for these drugs: substantial shifts for zidovudine and minor changes for adefovir and tenofovir. Thus, resistance seems to be multi-factorial and complex, whereby numerous mechanisms, such as inhibitor binding, removal of terminators by pyrophosphorolysis and/or ATP-dependent unblocking, and enzyme fitness all interact to influence susceptibility. Gaining insight into the mechanisms of resistance and mutational interactions may have clinical implications for patients who have extensive drug resistance mutations. Acknowledgements We thank Craig Gibbs, Mick Hitchcock and Damian McColl for helpful discussion and review of the manuscript, Melanie Lehwalder for administrative support, Manuel Tsiang for equation derivation and help with SigmaPlot Regressions, and Michael Parniak for the generous gift of recombinant RT mutants International Medical Press

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