ORIGINAL ARTICLE. Ó 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

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1 ORIGINAL ARTICLE Effect of polymorphisms on the replicative capacity of protease inhibitor-resistant HIV-1 variants under drug pressure C. Suñé 1, L. Brennan 1, D. R. Stover 2 and T. Klimkait 1,3 1 Basel Centre of HIV Research (BCHR), Institute for Medical Microbiology, Basel, Switzerland, 2 MDS Proteomics, Inc., Burlington, MA, USA and 3 InPheno AG, Basel, Switzerland ABSTRACT The role of drug pressure on the replicative capacity of protease inhibitor-resistant HIV-1 variants and the contribution of a common amino-acid polymorphism in the protease gene (L63P) to this process were investigated. Using HIV-1 variants resistant to the protease inhibitors saquinavir (G48V L90M) or indinavir (A71V V82T I84V), viral replication was studied in the presence or absence of inhibitor and a mutation at position 63. The initial changes diminished enzyme function of the protease and reduced replicative capacity for both virus mutants. Addition of the respective inhibitor blocked the wild-type, but was also able to delay the replication kinetics of either mutant, revealing the limits of resistance. Importantly, the polymorphic change L63P, although not conferring inhibitor resistance by itself, provided a significant replication benefit to both mutant viruses, particularly under drug pressure, and may reveal a far-reaching compensating power of polymorphic changes. This may drive evolution and the directed selection of protease inhibitor-resistant HIV-1 variants, a finding with significant clinical and diagnostic implications. Keywords HIV, protease inhibitors, polymorphisms, resistance, fitness Original Submission: 22 January 2003; Revised Submission: 10 May 2003; Accepted: 20 May 2003 Clin Microbiol Infect 2004; 10: INTRODUCTION Resistance is one of the important factors limiting drug efficacy against HIV-1 and can be directly linked to mutations at specific amino-acid positions of the reverse transcriptase (RT) and protease (PR) proteins, thereby allowing the virus to replicate in the presence of the respective inhibitors. The high number of replication events during HIV-1 infection in vivo (an estimated 10 8 )10 9 new virions are produced daily) [1,2], along with the lack of a proofreading function in the RT enzyme (producing more than one alteration/virus copy [3]), serves as a powerful driver of spontaneous genetic diversity in the HIV-1 population (variants, quasispecies) in the infected host. This mechanism potentially provides a random pool of viral variants, which can undergo selection for Corresponding author and reprint requests: T. Klimkait, Institute for Medical Microbiology, Petersplatz 10, 4003 Basel, Switzerland Tel: Fax: thomas.klimkait@unibas.ch drug resistance even during ongoing antiretroviral therapy. This claim is supported by the observation that a number of the currently known mutations associated with resistance to RT or PR inhibitors have been identified in virus isolates from drug-naive patients [4 9]. Recent studies correlate an increased occurrence of specific secondary mutations or polymorphic changes with elevated rates of clinical treatment failure [6,10,11]. Among the most common amino-acid polymorphisms in the HIV-1 PR gene is a leucine-to-proline substitution at residue 63 (L63P), which can occur in >50% of drug-naive patients [5]. Previous in-vitro studies have examined the impact of this mutation in the absence of inhibitor and have demonstrated that it improves replication of HIV-1 variants [12,13]. It has therefore been suggested that L63P may compensate for the deleterious effects that other resistanceconferring mutations can have on viral replication. At present, however, the influence of L63P on the replicative capacity of HIV-1 under drug pressure, a clinically more relevant situation during therapy, is unknown. Ó 2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 120 Clinical Microbiology and Infection, Volume 10 Number 2, February 2004 Mutations accumulated by HIV-1 PR inhibitor (PI)-resistant variants can significantly change the catalytic activity of the enzyme, and in many cases its enzymatic activity is diminished, which can compromise viral replication. Examples have been found where such a decrease in enzyme function was restored by other compensatory mutations in the PR gene, thereby improving replicative capacity [12 16]. To date, studies on HIV-1 replication have often been performed in the absence of drug, although the replicative capacities of HIV-1 variants can be affected as soon as drug pressure is applied [17]. In the present study, a defined in-vitro system was used to generate several clinically relevant PI-resistant variants in order to analyse the effect of specific drug-selected mutations on the replicative capacity of the virus in the presence or absence of drug. MATERIALS AND METHODS Cells The CD4 + HeLa-derived cell line SX22-1 contains an integrated copy of a silent lacz reporter gene placed under the control of a complete HIV-1 long-terminal repeat promoter. The reporter gene is activated upon expression of HIV-1 Tat protein and thereby allows viral gene expression to be normalised independently of HIV PR or RT activity in transient transfection experiments [18]. SX22-1 cells were grown in Dulbecco s modified Eagle s medium (Gibco, Paisley, UK) supplemented with fetal bovine serum (Gibco) 10% v v, penicillin streptomycin (Sigma, St Louis, MO, USA) to 100 U and 100 mg L, respectively, and 10 mm HEPES (Sigma). The lymphocytic CEM-SS cell line was grown in RPMI 1640 medium (Gibco) supplemented with fetal bovine serum 10% v v, penicillin streptomycin, 200 mm L-glutamine L (Sigma), and 50 mm b-mercaptoethanol (Sigma). Protease inhibitors Drug stocks were prepared from commercially available formulations, kindly provided by M. Battegay (Basel, Switzerland), by dissolving the drugs in water or dimethylsulphoxide to give 10 mm solutions which were stored at ) 20 C. Working solutions were prepared fresh in cell culture medium. Generation of molecular clones Relevant PR mutations were specifically introduced by PCRbased mutagenesis into a fully infectious pnl4-3 derivative with shortened cellular flanks, termed pnl-nf. This clone possesses a B-consensus PR sequence and carries a unique restriction site immediately downstream from the PR gene, such as SbfI. This modification does not change the efficiency of cleavage of the protease site or the overall replicative capacity of the new clone (L. Brennan, personal communication). For the simultaneous introduction of two or more mutations into the same clone, a sense oligonucleotide carrying the most distal mutation was used along with a reverse primer that hybridised downstream from the PR gene. PCR reactions were performed with Pwo polymerase (Roche Biochemicals, Mannheim, Germany). In order to bring another mutation into the protease gene, this PCR product itself then served as the reverse primer, along with a further mutant oligonucleotide mapping in the PR gene upstream from the first mutation. The product of such a second PCR was gel-purified and further extended with the same technique to reach a unique ApaI site in the Gag region of pnl-nf. The final PCR product was double-digested with ApaI SbfI and re-ligated into the complementing vector fragment of the proviral clone pnl-nf. After transformation into HB101 k, the alterations in the reconstituted HIV plasmid harbouring a mutated PR gene were verified by standard dideoxy sequencing on an ABI 310 capillary sequencer (Applied Biosystems, Warrington, UK). Cellular processing of pr55 Gag proteins For DNA transfection in 24-mm culture dishes, SX22-1 cells were first seeded in each well. On the following day, cells were transfected with 0.2 lg of proviral plasmid DNA of the respective pnl-nf derivatives using Lipofectamine 2000 (Invitrogen, Basel, Switzerland). After incubation for c. 48 h, the cell layers were scraped, pelleted by centrifugation and lysed in Nonidet-P40 0.5% v v, 10 mm Tris-HCl, ph 7.5. After a further brief centrifugation step, the supernatant was mixed with an equal volume of standard SDS-PAGE loading buffer, boiled for 5 min, electrophoretically separated on SDS-acrylamide 12% w v gels, and electro-transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). The resulting immunoblots were developed with a mouse anti- HIV-1 p24 monoclonal antibody (Ab ; gift of J. Mestan, Basel, Switzerland) and visualised by enhanced chemiluminescence as recommended by the manufacturer (Amersham, Little Chalfont, UK). Studies of viral replication kinetics and coculture experiments Purified plasmid DNA (0.2 g; protocol of Qiagen, Hilden, Germany) from HIV-1 clones was used to initiate virion production after transfection into SX22-1 cells. At 48 h posttransfection, defined mixes of Tat expression-normalised SX22-1 cells (as specified in the figure legends) were used to infect CEM-SS cells, each in the absence and presence of drug. Viral cultures were maintained by serially passaging them every 3 4 days in fresh, inhibitor-containing medium. Samples were collected and used to infect SX22-1 indicator cells. Virus production was quantified by measuring Tat-dependent b-galactosidase production with a colourimetric assay 48 h after infection, as described previously [18]. Sequencing of variant HIV-1 protease genes In summary, total RNA was isolated from infected CEM-SS cultures with a standard protocol (Qiagen) and subjected to

3 Suñé et al. Replicative capacity of PI-resistant HIV 121 cdna synthesis with protease-specific primers and MULV reverse transcriptase Superscript II (Invitrogen), used according to the manufacturer s specifications. Portions of these reverse transcriptase reactions were amplified by PCR in a 50-lL volume containing 200 lm datp, dgtp, dctp and dttp (Sigma), 1 lm of each primer, and 2 U of Hotstar DNA polymerase (Qiagen) in the manufacturer s reaction buffer. PCR products were then purified and inserted into the pgem- T vector (Promega, Madison, WI, USA). Competent HB-101 k cells were transformed with these plasmids and grown on LB agar plates containing ampicillin. A minimum of ten colonies was picked for each sample and plasmid DNA prepared (standard Qiagen plasmid procedure), followed by dideoxysequencing using HIV protease-specific primers. (kd) WT G48V/L90M A71V/V82T/I84V D25N Mock pr55 p24 Gag Gag RESULTS Cleavage of intracellular Gag protein in HIV-1 variants with protease mutations In order to examine the direct, selective influence of mutations on enzymatic activity and replicative capacity in the absence or presence of the corresponding drug, specific clonal PI-resistant variants of HIV-1 were engineered. The mutants selected were the saquinavir-resistant mutant G48V L90M [19], the indinavir-resistant mutant A71V V82T I84V [20] and, as a negative control, the inactive catalytic centre mutant D25N [21]. Plasmid DNA of either the complete, infectious wild-type (wt), or of isogenic variants carrying mutated protease genes, was transfected into SX22-1 cells. Viral protein processing was analysed with specific antibodies that recognise Gag precursor (pr55) as well as mature (p24) proteins in cell lysates (Fig. 1). Shifted ratios of pr55 to p24 in favour of the precursor were indicative of impaired HIV protease function. As shown in Fig. 1, levels of Gag processing similar to those of the wt were observed for the saquinavir-resistant G48V L90M mutant. In contrast, only greatly reduced levels of processed Gag were detectable for the indinavirresistant A71V V82T I84V mutant. Replicative capacities in the absence or presence of drug pressure: competition between HIV-1 variants Fig. 1. Processing of pr55 Gag in HeLa CD4 + cells transfected with an infectious HIV-1 plasmid carrying wild-type (wt) or mutated PR sequences as indicated. Lysates from day 2 were analysed for precursor (pr55 Gag ) and cleavage (p24 Gag ) proteins by immunoblotting. D25N is an inactive catalytic centre mutant with no pr55 processing. Molecular mass size markers (in kda) are shown on the left. Lysates from uninfected cells served as a negative control ( mock ). As the coexistence of several virus variants in a dynamic balance may be a typical in-vivo situation during drug therapy, replication was studied in co-culture competition experiments. This allowed the relative viral replication fitness of a mutant to be assessed against the wt by determining changes in their relative proportions after several cycles of replication. Mixes of wt and mutants at ratios of 1 : 1 and 10 : 1 were studied. The replication of the saquinavir-resistant G48V L90M mutant was first analysed parallel to or in combination with the wt, in either the presence or absence of drug (Fig. 2). At a saquinavir concentration of 10 nm, wt replication was suppressed (Fig. 2A), but replication kinetics (time to maximum) did not differ significantly between both viruses in the absence of drug (Fig. 2B). In competition experiments (Fig. 2C,D) with different wt mutant ratios in the absence of drug pressure, only wt sequences were identifiable by subsequent genotypic analysis (Fig. 3). The disappearance of G48V L90M indicated a superior replication capacity of the wt over this mutant in the absence of drug pressure, thereby suggesting that the cost of acquiring mutations associated with a high level of resistance to PIs may be a significant impairment of viral fitness. The fact that both mutants showed a similar efficiency of maturation of viral proteins (Fig. 1; additional data not shown) suggests that other compensatory mechanisms play a role. When saquinavir was present during virus propagation, only drug-resistant HIV-1 mutants were found in most of the clones analysed (Fig. 3). In 10 : 1 mixtures, three of the ten clones analysed had lost the inhibitor-associated mutation at position 48 (Fig. 3), and a spontaneous L63P change was seen

4 122 Clinical Microbiology and Infection, Volume 10 Number 2, February 2004 (A) Viral replication [arbitr. units] (C) Viral replication [arbitr. units] WT time [d] (B) time [d] time [d] G48V/L90M time [d] (D) WT:G48V/L90M (1:1) WT:G48V/L90M (10:1) no saquinavir Viral replication [arbitr. units] Viral replication [arbitr. units] 10 nm saquinavir Fig. 2. Replicative fitness of the G48V L90M variant in the absence (solid circles) or presence (open squares) of saquinavir. Using the integrated lacz reporter as indicated in Materials and Methods, normalised viral supernatants generated in transfected SX22-1 cells were used to initiate infections in CEM-SS cells. Replication profiles were obtained for wt (A), G48V L90M (B), or defined mixes at indicated ratios (C,D). Virus expression is expressed as cell-associated induction of the viral long-terminal repeat in arbitrary units (X-Gal conversion). Fig. 3. (A) Sequence comparison after serial passage of defined mixes of wt with the G48V L90M mutant in the presence of saquinavir in the experiments as depicted in Fig. 2. The number of clones with identical sequence is shown on the right (no. of clones). Amino-acid positions associated with drug resistance are highlighted in grey; dotted lines mark sequence identity. (B) Number of emerging identical clones from CEM-SS cell infections. Clones were sequenced on day 32 for (1) and (2), or on day 14 for (3). in one clone (Fig. 3). Similar changes were not detected in 1 : 1 mixtures of wt and mutant. The question of whether the new mutations L90M and G48V L63P L90M observed in this experiment reflected spontaneous mutations or DNA recombination events was not further investigated. The fact that only some of the 10 : 1 samples showed these alternative mutations argued against a PCR artifact. Nevertheless, the detection of new mutations may indicate a replicative advantage in the presence of drug. To test this possibility, CEM-SS cultures were infected in parallel with viral clones containing one of the variants L90M, G48V L90M, or L63P G48V L90M, with monitoring of intracellular pr55 Gag -precursor cleavage and viral replication (Fig. 4). The wt and L90M mutants showed indistinguishable Gag cleavage profiles, a finding which is compatible with a similar catalytic efficiency of both proteases, as reported previously [22]. In contrast, Gag processing in the G48V L90M double mutant was reproducibly delayed, in agreement with a reduced catalytic

5 Suñé et al. Replicative capacity of PI-resistant HIV 123 efficiency in a cell-free enzymatic system for protease activity (results not shown). Acquisition of the L63P substitution yielded a viral protease (G48V L63P L90M) permitting more efficient Gag cleavage when compared with the corresponding double mutant (G48V L90M) (Fig. 4A). When the relative viral replication in the absence of drug was assessed, the L90M mutant appeared to replicate with a slightly higher rate than the wt, and showed a saquinavir susceptibility identical to that of the wt virus (Fig. 4B,C). Competition experiments indicated that the wt and L90M had similar replication rates, since, after infection with a 1 : 1 mixture, seven of 13 recovered clones were wt and six were the L90M mutant (data not shown). In contrast, the G48V L90M double mutant showed delayed replication, but had increased resistance to saquinavir compared with the wt or the L90M-only mutant (Fig. 4B,C,D). In contrast, when a specific additional leucine-to-proline alteration was introduced at position 63, this correlated with increased viral fitness at all tested drug concentrations, without itself affecting drug resistance (Fig. 4D,E). DNA sequencing corroborated the identity and integrity of the respective viral species after completion of the culture period of 18 days. No additional, potentially drug-associated mutations were detected after cell passage (data not shown). In a similar way, the replication properties of the clonal indinavir-resistant mutants A71V V82T I84V and L63P A71V V82T I84V were analysed in the absence or presence of drug. The latter mutant was generated to study the potential impact of the L63P change in a different mutational context and for a different drug (Fig. 5). In the presence of 50 nm indinavir, both mutants replicated with markedly delayed kinetics compared to replication in the absence of indinavir (Fig. 5B,C), while virus sequencing (Fig. 3B) revealed the genetic integrity of the respective mutants under drug pressure. An indistinguishable replication profile for both mutants demonstrated that the only genetic difference, mutation L63P, did not provide any replication advantage when no drug was present (Fig. 5B,C), and sequencing of wt mutant mixes under this condition identified only wt sequences (Fig. 3B). Similar results were obtained in infections of either CEM-SS cells or primary peripheral blood Fig. 4. Replicative fitness of the protease variants L90M, G48V L90M and L63P G48V L90M in the absence and presence of drug, using cell-free virus infections of CEM-SS cells. (A) Virus expression was analysed on days 6, 10 and 14 postinfection with a p24 Gag -specific antibody. Precursor (pr55) and mature (p24) proteins are marked, and molecular size markers (kda) are shown on the left. (B E) Virus replication profiles for wt and mutants are shown as functions of time and were obtained in the absence (solid circles) or presence of the indicated saquinavir concentrations. Viral replication (in arbitrary units) was quantified via X-Gal conversion.

6 124 Clinical Microbiology and Infection, Volume 10 Number 2, February 2004 Fig. 5. Replicative fitness of the two indinavir-resistant variants in the absence (solid circles) or presence (open squares) of indinavir in a co-culture of expression-normalised, transfected human cells mixed with CEM-SS cells. Replication profiles of individual viruses (A C) or of 1 : 1 mixtures of wt with A71V V82T I84V (D) or with L63P A71V V82T I84V (E) are plotted as a function of time post co-cultivation. Virus replication was assessed via cell-associated X-Gal conversion. mononuclear cells (data not shown). In contrast, when the inhibitor was present during the infections, the L63P A71V V82T I84V mutant replicated with much higher efficiency than the A71V V82T I84V mutant, and shifted the peak of expression from > 32 days to day 17 (Fig. 5B,C). This finding demonstrated that, under drug pressure, the L63P change itself is able to partially restore the delayed replication kinetics of the A71V V82T I84V mutant, a finding that correlates with the role of L63P seen for the G48V L90M mutant. DISCUSSION Virus replication in the HIV-infected patient is influenced by a complex array of patient-specific factors driving immunological responses and drug metabolism, and by the potency of an antiretroviral therapy regimen. It has been shown that specific point mutations in the key enzymes of HIV correlate with a modulation of virus replicative capacity [14,15,23]. This is likely to reflect a penalty for the acquisition of resistance to a potent drug, and could help explain cases where sustained clinical benefit is observed despite the presence of resistance-associated mutations [24,25]. Since the presence of therapeutic drugs in the circulation is now the standard clinical situation, the current study investigated in-vitro HIV-1 infections under high, but not sterilising, drug pressure. The results demonstrated that resistance is not simply the all-or-none ability of a virus to replicate in the presence of inhibitor, but also has to involve virus replicative capacity. However, Mammano et al. [17] have shown an inverse link between HIV fitness and drug concentration. Ongoing virus replication in the patient is likely to be the basis for the establishment of sequence heterogeneity, and thereby mixed virus populations. Such generation of coexisting variants of HIV probably occurs preferentially in periods when the virus is least restricted by drug pressure, e.g., after drug failure, during therapy interruption, or in the non-compliant patient

7 Suñé et al. Replicative capacity of PI-resistant HIV 125 [26 28]. However, these situations would not yield just one new virus variant, so that each new generation of virions will enter a continuous selection process for optimal adaptation to the current (therapeutic) situation. Even the limited setting of this study was able to demonstrate the appearance and selection of novel therapy-relevant HIV variants with mutant proteases (Fig. 3). A new mutant with only an L90M substitution did not gain resistance compared to the wt, but it possessed a higher replicative capacity than the initial G48V L90M double mutant [19,29]. The precise reason remains to be elucidated, but altered protein folding and stability might have contributed. The second spontaneously occurring L63P mutation fully compensated for the impaired replication of the double G48V L90M mutation, but without permitting the resulting virus to replicate at higher drug concentrations. These data complement those on drug-free infections, for which it has been reported that L63P itself provides a replication improvement for several HIV-1 variants [12,13]. Although some investigators have not found any appreciable gain in the level of resistance for G48V L63P L90M [30], the frequent appearance of the identical mutations in different patients during suboptimal monotherapy with ritonavir in vivo [31], or during failing saquinavir treatment [32], may be explained by superior replication of the mutated virus. Extending the data to the indinavir ritonavir-resistant mutant A71V V82T I84V links a viral growth advantage more tightly to drug treatment; in the absence of drug, the L63P substitution failed to provide any replication advantage to the A71V V82T I84V-mutant (Fig. 5B,C), but it strongly enhanced viral replicative fitness in the presence of drug. These data correlate with the observed higher rate of HIV-1 replication in treated patients when the L63P substitution is present [12,33]. Although undetermined at this point, it is possible that the observed difference between the two model mutants G48V L90M and A71V V82T I84V with regard to their replication in the absence or presence of drug could be caused by the fact that a severe functional impairment of protease (e.g., in the A71V V82T I84V situation) could make the mutant enzyme the replication rate-limiting step, thus slowing virus replication even without any drug pressure being applied. In summary, investigation of in-vitro replication of PI-resistant HIV-1 mutants under therapeutic drug pressure is suitable for assessing viral replicative fitness in the presence of therapeutic drugs, and could be of critical clinical relevance. It is possible to characterise viral parameters that may be useful for improving the prediction of success of a therapeutic regimen, which in turn could influence decisions about continuing or changing regimens [34,35]. Extended studies on a broader range of virus mutants will be required to link in-vitro data on specific mutational resistance patterns and viral fitness with therapeutic failure and patient outcome. ACKNOWLEDGMENTS We express our gratitude to Gaby Schaub and Claudia Joerg- Ettisberger for excellent technical expertise. We thank Manuel Battegay, Africa Holguin and Cristina Hernández-Munaín for critical review of the manuscript. This research was supported by a grant (no ) from the Swiss National Science Foundation. REFERENCES 1. Ho DD, Neumann AD, Perelson AS et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995; 373: Wei X, Ghosh SK, Taylor ME et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995; 373: Preston BD, Poiesz BJ, Loeb LA. Fidelity of HIV-1 reverse transcriptase. Science 1988; 242: Najera I, Holguin A, Quiñones-Mateu ME et al. Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy. J Virol 1995; 69: Kozal MJ, Shah N, Shen N et al. Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays. Nat Med 1996; 2: Perno CF, Cozzi-Lepri A, Balotta C et al. Secondary mutations in the protease region of human immunodeficiency virus and virologic failure in drug-naive patients treated with protease inhibitor-based therapy. J Infect Dis 2001; 184: Lech WJ, Wang G, Li Yan YL et al. In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects. J Virol 1996; 70: Pieniazek D, Rayfield M, Hu DJ et al. Protease sequences from HIV-1 group M subtypes A H reveal distinct amino acid mutation patterns associated with protease resistance in protease-naive individuals worldwide. AIDS 2000; 14: Cane PA, de Ruiter A, Rice P et al. Resistance-associated mutations in the human immunodeficiency virus type 1 subtype C protease gene from treated and untreated

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