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JAIDS Journal of Acquired Immune Deficiency Syndromes:
doi: 10.1097/QAI.0b013e3181fe9450
Basic and Translational Science

Reduced Replication Capacity of NL4-3 Recombinant Viruses Encoding Reverse Transcriptase–Integrase Sequences From HIV-1 Elite Controllers

Brumme, Zabrina L PhD*†‡; Li, Chun BSc‡§; Miura, Toshiyuki MD‡‖; Sela, Jennifer BSc; Rosato, Pamela C BSc‡; Brumme, Chanson J BSc†‡; Markle, Tristan J BSc*; Martin, Eric BSc*§§; Block, Brian L BSc‡; Trocha, Alicja DVM‡; Kadie, Carl M PhD¶; Allen, Todd M PhD‡; Pereyra, Florencia MD‡; Heckerman, David MD, PhD¶; Walker, Bruce D MD‡#; Brockman, Mark A PhD*§§†‡

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From the *Faculty of Health Sciences and §§Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby BC, Canada; †British Columbia Centre for Excellence in HIV/AIDS, Vancouver BC, Canada; ‡Ragon Institute of MGH, MIT, and Harvard, Boston, MA; §Program of Biological Sciences in Dental Medicine, Harvard University, Cambridge, MA; ∥Division of Infectious Diseases Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan; ¶Microsoft Research, Redmond WA; and #Howard Hughes Medical Institute, Chevy Chase, MD.

Received for publication January 5, 2010; accepted September 23, 2010.

Supported in part by grants AI028568 and AI030914 from the NIAID-NIH, the Howard Hughes Medical Institute, the Harvard University Center for AIDS Research, the Bill and Melinda Gates Foundation, and a gift from the Mark and Lisa Schwartz Foundation (to B.D.W.), and an Operating Grant from the Canadian Institutes of Health Research (CIHR) and a Jim Gray Seed Grant from Microsoft Research (to M.A.B. and Z.L.B.). Z.L.B. is supported by a CIHR New Investigator Award. C.J.B. is supported by a NSERC Julie Payette Scholarship.

Presented in part as: Li C, Brumme ZL, Miura T, Rosato P, Sela J, Brumme CJ, Heckerman D, Pereyra F, Walker BD, Brockman MA. Reduced replication capacity of NL4-3 chimeric viruses encoding reverse transcriptase-integrase sequences from HIV-1 elite controllers. AIDS Vaccine. 2009. Abstract P09-11.

The authors Z.L.B., C.L. contributed equally.

The authors declare no conflicts of interest related to this study.

Correspondence to: Mark A. Brockman, PhD, Associate Professor, Molecular Biology and Biochemistry, Simon Fraser University, South Sciences Building, Room 7153, 8888 University Drive, Burnaby, BC, Canada V5A 1S6 (e-mail: mark_brockman@sfu.ca).

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions this article on the journal's Web site (www.jaids.com).

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Abstract

Background: Identifying viral and host determinants of HIV-1 elite control may help inform novel therapeutic and/or vaccination strategies. Previously, we observed decreased replication capacity in controller-derived viruses suggesting that fitness consequences of human leukocyte antigen (HLA) class I-associated escape mutations in Gag may contribute to this phenotype. This study examines whether similar functional defects occur in Pol proteins of elite controllers.

Methods: Recombinant NL4-3 viruses encoding plasma RNA-derived reverse transcriptase-integrase sequences from 58 elite controllers and 50 untreated chronic progressors were constructed, and replication capacity measured in vitro using a green fluorescent protein (GFP) reporter T-cell assay. Sequences were analyzed for drug resistance and HLA-associated viral polymorphisms.

Results: Controller-derived viruses displayed significantly lower replication capacity compared with those from progressors (P < 0.0001). Among controllers, the most attenuated viruses were generated from individuals expressing HLA-B*57 or B*51. In viruses from B*57+ progressors (n = 8), a significant inverse correlation was observed between B*57-associated reverse transcriptase-integrase escape mutations and replication capacity (R = −0.89; P = 0.003); a similar trend was observed in B*57+ controller-derived viruses (n = 20, R = −0.36; P = 0.08).

Conclusions: HIV-1 Pol function seemed to be compromised in elite controllers. As observed previously for Gag, HLA-associated immune pressure in Pol may contribute to viral attenuation and subsequent control of viremia.

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INTRODUCTION

Elite controllers are a rare group of HIV-1-infected individuals who spontaneously maintain plasma viremia below the limit of standard clinical detection (<50 viral RNA copies/mL) without antiviral therapy.1 Elucidating the mechanisms responsible for this phenotype may reveal host and viral factors that may be modulated for prophylactic or therapeutic intervention.

HIV-1 replication capacity (RC) likely plays an important role in pathogenesis,2,3 but its relevance to the elite controller phenotype remains unclear. Although a recent examination of a large number of elite controller-derived HIV sequences revealed no evidence of gross mutational defects, large insertions or deletions, nor shared ancestry,4 virus function may nevertheless be compromised. Indeed, previous studies have reported lower RC in viruses isolated from viremic long-term nonprogressors compared with those from chronically infected individuals,2,3,5 and reduced entry efficiency has been observed for elite controller-derived envelopes compared with those from chronically infected individuals.6

Although RC is determined in large part by the founder virus acquired at transmission,7,8 it can change over time as host and other selective pressures drive intrahost HIV-1 evolution.9-11 In vitro fitness costs of HLA-restricted cytotoxic T-lymphocyte (CTL) escape mutations in Gag12-17 and Nef18 have been demonstrated, and evidence suggests that immune-mediated fitness defects may be relevant to the controller phenotype.19,20 A recent case report described reduced RC of virus isolated from a B*27/B*57-expressing elite controller compared with the transmitted donor virus.19 Furthermore, we have previously described reduced in vitro RC of recombinant viruses expressing Gag-Protease from elite controllers compared with progressors in chronic20 and acute/early21 infection, an observation attributable at least in part to immune selection.20,22 A biologically relevant role for immune-mediated fitness defects is supported by relative early viremia control in individuals who acquire HIV-1 harboring escape mutations from donors expressing protective HLA alleles.7,8 Moreover, evidence for sequential reductions in RC as a result of the accumulation of HLA-restricted CTL escape mutations has been reported in Gag.16,23

However, comparably little is known about the consequence of mutations outside of the Gag gene on viral RC in elite controllers, and what relevance this may have to the controller phenotype. Because mutations in the Pol gene that emerge under antiretroviral drug selection pressures can affect fitness,24-28 we therefore examined whether elite controller viruses exhibited functional defects in this gene. To do this, recombinant viruses encoding plasma RNA-derived reverse transcriptase (RT)-integrase sequences were generated in an NL4-3 virus backbone from 58 elite controllers and 50 untreated chronic progressors and their in vitro RC was examined using a GFP reporter T-cell assay.20,29 Similar to previous observations for Gag-Protease20,21 and Envelope,6 we observed reduced function of controller-derived RT-integrase, indicating that differences in Pol activity may contribute to HIV-1 control.

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METHODS

Study Participants

Fifty-eight elite controllers {all <50 copies RNA/mL; median CD4 = 799 [interquartile range (IQR): 593-1037] cells/mm3} and 50 untreated chronic progressors [median viral load 4.98 (IQR: 4.51-5.35) log10 copies RNA/ml; median CD4 = 318 (IQR: 61-476) cells/mm3] were included. Characteristics of this elite controller cohort have been described previously.30 In addition, 76 of these 108 (70%) patients were previously evaluated for Gag-Protease function.20 HLA class I typing was performed using standard sequence-based methods. This study was approved by the institutional review board at Massachusetts General Hospital; written informed consent was obtained from all participants.

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Generation of Recombinant RT-Integrase Viruses
Bulk (Quasispecies) Method

For elite controllers, reverse transcriptase-polymerase chain reaction (RT-PCR) products spanning Pol were generated as described.4 For progressors, HIV-1 Pol was RT-PCR amplified from extracted plasma HIV RNA using sequence-specific primers. Second-round polymerase chain reaction (PCR) was performed using PAGE-purified “recombination primers” designed to match the NL4-3 sequence directly upstream of RT (forward; 100bp) and downstream of integrase (reverse; 98bp). Primer sequences are available upon request.

Plasmid pNL4-3▵RT-Integrase was developed by inserting unique restriction enzyme sites for BstEII at the 5′ end of RT and the 3′ end of integrase using the QuikChange XL kit (Stratagene) followed by deletion of the intervening region by BstEII digestion (New England Biolabs, Ipswich, MA). This plasmid was maintained using Stbl3 E. coli cells (Invitrogen, Burlington, ON, Canada). To generate recombinant viruses, 10 μg of BstEII-linearized plasmid plus 50 μL of second-round amplicon (approximately 5 μg) were mixed with 2.0 × 106 cells of a Tat-driven GFP reporter T-cell line (GXR25 cells29) in 800 μL of R10+ medium (RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin), and transfected by electroporation using a BioRad GenePulser Xcell (exponential protocol: 300V, 500 μF). After transfection, cells were rested for 45 minutes at room temperature, transferred to 25 cm2 flasks in 5 mL of R10+ medium, and fed with 5 mL R10+ medium on day 5. GFP expression was monitored daily by flow cytometry (FACScalibur, BD Biosciences) starting on day 10. Once GFP+ expression reached ∼15% among viable cells, representing the early phase of exponential spread, culture supernatants containing the recombinant viruses were harvested and aliquots stored at −80°C.

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Clonal Method

In addition, RT-integrase sequences from a random subset of 14 controllers and 10 progressors were cloned (TOPO-TA cloning kit; Invitrogen, Burlington, ON, Canada), purified and used as starting material to generate recombinant viruses as described above. All clones were resequenced to confirm patient origin.

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RC Assays

Virus titers and replication assays were performed as described.12,20,29 Replication assays were initiated at multiplicity of infection = 0.003, and included 6 negative (uninfected cells only) and 6 NL4-3 controls in each experiment. For each virus, the natural log of the slope of the percent of GFP+ cells was calculated during the exponential phase of viral spread in culture (days 3-6). This value was divided by the mean rate of spread of NL4-3 to generate a normalized, quantitative measure of RC. An RC of 1.0 indicates a rate of spread equal to NL4-3, whereas RC <1.0 and >1.0 indicate rates of spread that are slower than or faster than NL4-3, respectively. All assays were performed in triplicate in independent experiments, and replication rates were averaged. The experimental procedure is illustrated in the Supplemental Material (see Figure 1 Supplemental Digital Content 1, http://links.lww.com/QAI/A109).

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Resequencing of Recombinant Viral Stocks

For all bulk (quasispecies-containing) recombinant viruses, HIV-1 RNA was extracted from viral culture supernatant (QIAamp viral RNA kit; Qiagen), amplified and sequenced as described in4 and compared to the original plasma HIV-1 RNA sequences (Fig. 1). All viruses were confirmed as subtype B using RIP (http://www.hiv.lanl.gov). Nucleotide alignments were performed using GeneCutter and maximum-likelihood phylogenetic trees were generated using PHYml31 (both available at http://www.hiv.lanl.gov). Trees were visualized using Figtree v.1.2.2 (http://tree.bio.ed.ac.uk/software/figtree/). Resistance mutations were identified using the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu/). Most controller-derived RT-Integrase sequences were previously deposited in Genbank.4 Accession numbers for the remaining sequences from controllers and progressors are GQ284657-GQ284730.

Figure 1
Figure 1
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Statistical Analysis

Student T test was used to compare differences in RC between groups (eg, controllers/progressors; presence/absence of HLA alleles, etc). Spearman and Pearson correlation was used to investigate the relationship between clinical parameters (CD4/pVL) and the presence of HLA-associated escape mutations, respectively, and viral RC. In an exploratory analysis, the Mann-Whitney U Test was used to identify specific amino acids in RT-integrase associated with RC; here, Q values were used to address multiple tests.32

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RESULTS

Generation of Recombinant Viruses Expressing RT-Integrase Sequences From Controllers and Progressors

Recombinant viruses were generated using bulk patient plasma-derived PCR amplicons containing RT and integrase sequences and stocks harvested during the early phase of exponential viral spread as described in Methods. For elite controllers (n = 58), the median time to harvest was 25 days (IQR: 21-28), whereas for progressors (n = 50), it was 19 days (IQR: 15-21). This difference was statistically significant (P < 0.0001). The RT-integrase sequences of the recombinant virus and the original plasma HIV RNA were highly concordant (Fig. 1). The median number of full amino acid differences between plasma and recombinant sequences was 1 (IQR: 0-2) of a total of 849 codons spanning RT-integrase, a similarity of 99.9%. In a more conservative analysis where amino acid mixtures were considered full differences, the median number of differences was 6 (IQR: 3-9) (99.3% similarity); values that are comparable to the average interlaboratory nucleotide concordance of sequence-based genotypic drug resistance assays (99.4%).33 These data indicate that our approach did not result in substantial in vitro selection and that at least some quasispecies diversity was maintained in the recombinant viral stocks.

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Reduced RC of RT-Integrase Viruses Derived From Elite Controllers

RC of bulk (quasispecies-containing) recombinants was assayed in 3 independent experiments and results reported as the mean. Concordance between replicates was high [R = 0.77, P < 0.0001; with a median difference between replicates of 8.03% (IQR: 3.76%-16.4%)].

Controller-derived RT-integrase recombinant viruses constructed using bulk (quasispecies-containing) methods displayed significantly reduced RC compared with those derived from progressors (Figs. 2A, B). The median RC of controller viruses was 0.83 (IQR: 0.63-0.96) compared with 0.98 (0.89-1.07) for progressor viruses (P < 0.0001). To assess whether this result may be affected by the diversity of the recombinant quasispecies, we stratified recombinant virus sequences based on the presence or absence of amino acid mixtures in the RT-integrase region, but observed no significant differences in RC between the 2 in either controllers or progressors (Fig. 2C). The overall differences in RC between controllers and progressors remained highly statistically significant regardless of the presence or absence of amino acid mixtures (ANOVA P < 0.0001).

Figure 2
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To further rule out potential bias due to the quantity and/or diversity of the initial bulk PCR inoculum between controllers and progressors, and the potential existence of minority quasispecies in the recombinant virus stocks, we generated clonal recombinant viruses from a randomly selected subset of 14 controllers and 10 progressors. Consistent with the original findings, median RC of clonal controller viruses was 0.73 (IQR: 0.61-0.98) compared with 0.97 (0.86-1.08) in the progressors (P = 0.028; Figure 2A, Supplemental Digital Content 2, http://links.lww.com/QAI/A110). In addition, robust concordance was observed between RC of independently constructed clones (n = 10 pairs, R = 0.84, P = 0.002, not shown) and between the RC of quasispecies-containing versus clonal recombinant viruses (R = 0.7, P = 0.0003, Figure 2B, Supplemental Digital Content 2, http://links.lww.com/QAI/A110).

Taken together, the RT-integrase region from most controllers exhibited reduced function compared with progressors, regardless of whether quasispecies-containing or clonal recombinants were evaluated.

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Reduced RC Is Not Due to Resistance Mutations

Resistance mutations can affect viral RC.24-28 Although all patients were untreated at the time of sample collection, 8 of 58 (14%) controller-derived and 8 of 50 (16%) progressor-derived viruses harbored at least 1 major resistance mutation in RT (P = 0.8), most frequently at codons 215 (n = 9) and 219 (n = 6), K70R (n = 5), D67N (n = 4), and others. Of these 16 sequences, 10 encoded ≥2 resistance mutations. After exclusion of the 16 resistant viruses from analysis, the difference in RC between controller and progressor-derived viruses remained statistically significant (P < 0.0001, not shown). Furthermore, no significant differences were observed between resistant and nonresistant viruses within each patient group (P > 0.05), suggesting that decreased RC in controllers is not due to drug resistance mutations.

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Reduced RC Does Not Correlate With Clinical Parameters, or Gag/Protease Function, in Controllers or Progressors

We next addressed whether RT-integrase function correlated with clinical parameters. Among controllers, all pVL were <50 copies/mL, and the median CD4 count was 799 (IQR: 593-1037) cells/mm3. Among progressors, the median pVL was 4.98 (IQR: 4.51-5.35) log10 HIV RNA copies/mL, and the median CD4 count was 318 (IQR: 61-476) cells/mm3. In an analysis stratified by patient group, we observed correlation neither between RC and CD4 count in controllers (Spearman R = −0.03, P = 0.8) or progressors (R = 0.06, P = 0.7) nor between replication and pVL in progressors (R = 0.05, P = 0.7) (not shown).

We also evaluated whether RT-integrase RC correlated with functional RC data for Gag-Protease previously obtained on a subset of these individuals (reported in20). Although both the previous and present study report reduced RC among controller-derived viruses, we observed no correlation between RC of Gag-Protease viruses and RT-integrase viruses in an analysis stratified by patient group [controllers (n = 38, R = −0.09, P = 0.6); progressors (n = 38, R = 0.13, P = 0.4)], suggesting that these observations are largely independent.

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Associations Between RC and HLA Class I Expression

We next investigated associations between RC and HLA class I allele expression, stratified by patient group (Fig. 3). In controllers, no statistically significant associations were observed, however, the poorest replicating viruses originated from HLA-B*51- and B*57-expressing individuals (P = 0.09 and P = 0.08, respectively). Among progressors, viruses from A*01-expressing and C*02-expressing individuals displayed significantly lower RC (P = 0.016 and P = 0.02, respectively).

Figure 3
Figure 3
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Correlation Between RC and HLA-Associated Sequence Polymorphisms

To further assess the impact of immune selection pressure on RT-integrase function, we investigated potential correlations between the presence of HLA-associated polymorphisms (defined according to a population-based analysis of >1500 subtype-B-infected individuals worldwide34) and RC. For each patient, we determined the total possible number of HLA-associated polymorphic sites in RT-integrase according to their HLA class I profile and the number of these sites that exhibited a known HLA-associated polymorphism in the recombinant virus sequence. No significant correlation was observed between RC and the overall burden of HLA-A, B, and C-associated polymorphisms in RT-Integrase assessed in absolute terms (“number of escaped sites”) or relative terms (“proportion of escaped sites”) in either controllers or progressors (all P > 0.05, not shown).

We next investigated the relationship between HLA-associated polymorphisms and RC in an allele-specific manner, for all alleles with frequencies ≥5. Although no correlations achieved statistical significance in controllers, an inverse relationship was observed between the number of B*57-associated polymorphisms and lower RC in B*57-expressing controllers (n = 20; R = −0.36, P = 0.1) (Fig. 4A). In B*57-expressing progressors, a strong inverse relationship was observed between the number of B*57-associated RT-integrase polymorphisms and RC (n = 8, R = −0.89, P = 0.003); no other significant correlations were observed (Fig. 4B). Therefore, although the average RC of viruses derived from B*57-expressing progressors did not significantly differ from the population average, the strong dose-dependent relationship between B*57-associated polymorphisms and decreasing RC suggests that these polymorphisms negatively influence RT-integrase function in a cumulative manner.

Figure 4
Figure 4
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No Association Between RC and Transmitted HLA-Associated Polymorphisms

Immune-mediated fitness defects have been reported in individuals acquiring HIV-1 from donors who express protective HLA alleles.7,8 Therefore, we investigated whether RC correlated with protective allele-associated viral polymorphisms in individuals not expressing these alleles. No significant difference was observed in the average number of polymorphisms associated with protective HLA alleles (defined as HLA-B*13, B*27, B*51, B*57, B*5801, and B*8135-40) in controllers (n = 19; mean protective allele-associated polymorphisms/sequence = 8.6) versus progressors (n = 22; mean polymorphisms/sequence = 7.6; P = 0.25) not expressing these alleles. Moreover, no correlation was observed between the number of protective HLA-associated polymorphisms and RC in controllers (R = 0.23, P = 0.34) or progressors (R = 0.34, P = 0.13) not expressing these alleles. An analysis restricted to B*57-associated mutations in controllers and progressors not expressing this allele also failed to demonstrate significant differences in polymorphism frequency between groups, nor correlations with RC (all P > 0.1, not shown). Altogether, these results suggest that the observed reductions in RC seen in controllers were not likely due to transmitted immune escape mutations in RT-integrase.

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Associations Between RC and Specific Amino Acid Residues in RT-Integrase

In an exploratory analysis, the Mann-Whitney U test was used to examine associations between RT-integrase amino acid variation and RC. This analysis was performed on the entire dataset, as well as stratified by patient group. In the combined analysis of all polymorphisms occurring at frequencies ≥5, 28 residues (20 in RT, 8 in integrase) were associated with RC at P < 0.05 (corresponding Q values 0-0.77; Table 1). The strongest association was observed for integrase codon 265, where the consensus Alanine was associated with significantly higher RC than the polymorphism Valine (P < 0.0001, Q = 0). Stratification by patient group also identified the codon 265 association as significant in controllers (P < 0.01, Q = 0.4) but not in progressors (not shown).

Table 1
Table 1
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DISCUSSION

We recently reported that elite controllers display defects in Gag-Protease function due in part to immune selection by protective HLA alleles, most notably B*57,20 thus further supporting an impact of Gag escape mutations on viral fitness and HIV disease.12-17,19,23 However, comparatively little is known about potential immune-mediated attenuation of other HIV-1 proteins and whether this might be relevant to the controller phenotype.

Here, we extend our previous findings and demonstrate that recombinant viruses encoding RT-integrase sequences derived from elite controllers displayed significantly reduced RC compared with viruses derived from untreated chronic progressors. This was true regardless of whether recombinant viruses captured quasispecies diversity or whether they were generated from cloned sequences. The observed defects in controller-derived viruses were not likely due to the presence of drug resistance mutations24-28 nor the presence of putative transmitted immune escape mutations,7,8 although the possibility that such mutations were transmitted but then subsequently reverted cannot be ruled out. The presence of RT drug resistance mutations in a minority of elite controller samples merits mention: previous treatment is not an exclusion criterion for the International HIV controllers study (http://www.hivcontrollers.org/). Alternatively, these mutations may represent transmitted resistance mutations.

Instead, reduced RC of controller-derived recombinant RT-integrase viruses may be explained, at least in part, by the selection of fitness-attenuating mutations that result from effective HLA-restricted CTL responses to the incoming virus. We observed that viruses generated from B*51 and B*57-expressing controllers exhibited the most profound RC defects. Both of these alleles restrict strong CTL responses against epitopes in Pol41,42 and have been identified as “protective” with respect to HIV disease progression.36,43-45 Furthermore, among B*57-expressing individuals, the number of B*57-associated polymorphisms correlated inversely with RC, suggesting that the accumulation of B*57-associated mutations in Pol can negatively impact viral RC in a dose-dependent manner. Similar results have been reported previously for HLA-B*5703-selected mutations in the Gag p24/capsid protein.16 Notably, although both B*57+ controllers and progressors harbored B*57-associated escape mutations, viruses from B*57-expressing controllers displayed further reduced RC than their progressor counterparts, suggesting the presence of additional functional defects in controller sequences beyond those associated with commonly observed primary escape mutations. Previous studies have described rare and/or unique Gag escape mutations associated with fitness costs in controllers,22 although nonimmune mechanisms cannot be ruled out. The selection of compensatory mutations in progressors could also contribute to observed differences, as has been reported in Gag.13 Observed associations between HLA-A*01 and C*02 and viral RC are also notable and merit further study.

Substantial overlap in the RC distribution of controller-derived and progressor-derived viruses indicates that reduced RT-integrase function is not common to all controllers. Similarly, viruses generated from B*51 and B*57-expressing progressors did not exhibit significantly reduced RC compared with their non-B*51/B*57-derived counterparts. This underscores the observation that expression of a protective allele does not guarantee viral attenuation, and that analysis at the individual sequence level is necessary to elucidate relationships between viral polymorphisms and fitness (indeed, analysis at the sequence-level revealed a significant dose-dependent relationship between the number of B*57-associated escape mutations and RC in B*57+ individuals). Furthermore, more than half of controllers expressed neither B*51 nor B*57, yet they still harbored viruses with reduced RC compared with progressors, indicating that immune selection pressure by these 2 alleles does not solely account for the observed effects. We can neither rule out transmission of attenuated viruses in at least some controllers nor the selection of unique mutations, immune-mediated or otherwise that incur fitness costs.

The inherent challenges associated with identifying elite controllers and extracting HIV RNA from individuals with undetectable plasma viral loads limit the size, and thus the power to comprehensively evaluate associations between RC and specific HLA alleles or viral polymorphisms. Despite this, we were able to identify a number of Pol codons that may be associated with reduced RC, including, among others, the B*57-associated V245E mutation in RT (residue 2 of B*57 ISW9 epitope46,47) that is selected relatively rapidly after infection in B*57 (and B*58)-expressing individuals.48 The observation that V245E reverts after transmission to non-B*57/B*58-expressing individuals48,49 also supports a fitness cost. Integrase 265V was identified as the strongest correlate of lower RC, but mechanisms for this are unclear. This residue lies within described B*15 and B*42-restricted epitopes; however, to our knowledge no HLA-associated polymorphisms have been reported at this position. Although candidate residues were identified in this exploratory analysis, a comprehensive identification of codons associated with Pol RC will require a larger dataset followed by in vitro validation.

In summary, we observed significantly reduced RC of recombinant NL4-3 viruses encoding RT-integrase from elite controllers compared with those from untreated chronic progressors, regardless of whether recombinant viruses captured quasispecies diversity or were generated from cloned sequences. Controller-derived viruses from individuals expressing “protective” HLA-B*51 and B*57 alleles exhibited the lowest overall RC, and a dose-dependent inverse relationship was observed between the number of B*57-associated mutations and viral replication in both controllers and progressors. Although it is well established that envelope is a major determinant of fitness,2,50 a growing body of evidence indicates that mutations outside envelope mediated by immune14,16,20,23 or other27 selective pressures may also result in fitness defects. Results are consistent with functional defects in viruses isolated from HIV-1 elite controllers,6,20,21 which may arise as a result of immune selection pressures that reduce viral RC.20

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ACKNOWLEDGMENTS

We gratefully acknowledge the efforts of the clinical and laboratory staff of the International HIV Controllers Study (IHCS), as well as the healthcare professionals and researchers who have referred HIV controllers or contributed samples to the IHCS. A complete list of contributors and collaborators can be found at http://www.hivcontrollers.org. We also thank the dedicated individuals who have participated in this study, without whom this research would not be possible. We also thank Aleksandar Filiposki at Simon Fraser University for laboratory assistance.

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Keywords:

Cytotoxic T-lymphocyte; elite controller; HIV-1; HLA class I; immune escape; Pol; viral replication capacity

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