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R5 to X4 Switch of the Predominant HIV-1 Population in Cellular Reservoirs During Effective Highly Active Antiretroviral Therapy
 
 
  JAIDS Journal of Acquired Immune Deficiency Syndromes
1 April 2005
 
Delobel, Pierre* ; Sandres-SaunŽ, Karine PhD*; Cazabat, Michelle*; Pasquier, Christophe MD, PhD*; Marchou, Bruno MD ; Massip, Patrice MD ; Izopet, Jacques PhD*
 
From *The Laboratory of Virology, and  The Department of Infectious Diseases, Purpan University Hospital, Toulouse, France.
 
Abstract
Summary: HIV-1 coreceptor usage plays a critical role for virus tropism and pathogenesis. A switch from CCR5 to CXCR4-using viruses can occur in the natural course of infection and correlates with subsequent disease progression. To investigate whether HIV-1 genetic evolution might lead to changes in virus coreceptor usage during highly active antiretroviral therapy (HAART), a longitudinal genotypic analysis of the virus found in cellular reservoirs was conducted in 32 patients with undetectable viral loads on HAART for 5 years. The genotype of the 3rd variable region of the env gene predicting coreceptor usage was retrospectively determined in the plasma or in peripheral blood mononuclear cells (PBMC) at baseline and then in PBMCs at months 30 and 60 of HAART. There was a switch from R5 to X4 variants in 11 of the 23 patients who harbored a majority virus population of R5 variants at baseline. X4 variants remained predominant in the 9 patients who harbored mainly X4 variants at baseline. The patients harboring predominantly X4 variants during HAART, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously a majority population of R5 variants. These results suggest that potent antiretroviral therapy produces the conditions necessary for the gradual emergence of X4 variants in cellular reservoirs.
 
In our study, clonal analysis of HIV-1 quasipecies performed in patients for whom an R5 to X4 switch occurred in the majority virus population during HAART (R5/X4 group) revealed that minority virus populations of X4 variants were usually present within the virus quasispecies at baseline. Phylogenetic analysis revealed that the emergent X4 variants on HAART were related to these minority species present in the plasma or PBMCs at baseline. The presence of minority virus populations of pathogenic X4 variants might also explain that baseline CD4+ T-cell counts were as low in the R5/X4 group as they were in the X4/X4 group. These findings suggest that HAART may produce the conditions necessary for the gradual selection of preexisting minor X4 variants in cellular reservoirs but do not prefigure whether this selection occurs passively through the progressive selection of latently X4-infected long-lived cells or actively through the preferential residual replication of X4 variants during HAART.
 
AUTHOR DISCUSSION
In the present study, we retrospectively investigated the evolution of the V3 region of HIV-1 env and its impact on coreceptor usage in 32 patients on prolonged suppressive HAART for 5 years. Three groups of patients can be described according to the coreceptor usage evolution of the majority virus population during HAART. The first group (n = 12) harbored a majority virus population of R5 variants at baseline that remained predominant throughout follow-up (R5/R5 group). The second group (n = 11) also harbored a majority virus population of R5 variants at baseline, but an R5 to X4 switch occurred during HAART (R5/X4 group). The 3rd group (n = 9) harbored a majority virus population of X4 variants at baseline that remained predominant during HAART (X4/X4 group).
 
Few previous studies have addressed the impact of antiretroviral therapy on virus tropism in various clinical settings. A recent study found an unexpectedly high frequency of X4 variants among treated patients infected with HIV-1 subtype C,32 although several previous reports had shown that subtype C viruses overwhelmingly use the CCR5 coreceptor in untreated patients at various stages of disease, including advanced AIDS.33,34 These findings suggest that antiretroviral therapy may have promoted the emergence of X4 variants in these patients. In contrast, X4 variants were found to be preferentially suppressed in some patients after the initiation of antiretroviral therapy. A reversion from SI to NSI variants was first reported in studies in which only low-potency antiretroviral regimens were used, resulting in an incomplete virologic response.35-37 More recently, the preferential suppression of X4 variants was also reported in some patients after the initiation of protease inhibitor-containing antiretroviral combination therapy.38-40 However, the reversion from X4 to R5 variants was generally observed shortly after the initiation of antiretroviral therapy in these studies, before the complete suppression of plasma viremia. Subsequent analysis revealed that the X4 virus component was suppressed transiently and that X4 variants usually reemerged after a variable delay.39,40 Consistent with these findings, both R5 and X4 variants have been found to persist in the latent reservoir during prolonged suppressive HAART.41,42 In our study, long-term follow-up was required to detect the R5 to X4 switch of the majority virus population in cellular reservoirs, and we cannot exclude that, in patients harboring X4 variants at baseline, a transient suppression of the X4 variants could have occurred at the initiation of HAART (as was probably the case for patient 26), followed by their subsequent reemergence.
 
The general assumption is that an accumulation of mutations in V3 is required to achieve the transition from R5 to X4 variants and that these mutations occur only during active virus replication in the absence of antiretroviral therapy.43 Nevertheless, we observed in the present study an R5 to X4 switch of the predominant virus population in the setting of effective HAART. We can speculate that this emergence of X4 variants on HAART is due to the preferential implication of X4 variants in cellular reservoirs, either through a progressive replenishment of the pool of resting memory CD4+ T cells by cells derived from long-lived naive CD4+ T cells harboring archival X4 species, or through the preferential residual replication of CXCR4-using viruses on HAART.
 
The restoration of CD4+ T cells in response to effective HAART depends mainly on a slow increase in naive CD4+ T cells.21,22 This subset represents a target cell type for HIV-1 but is usually infected at a lower frequency than memory CD4+ T cells.44 However, a trend toward equalization of viral loads in memory and naive CD4+ T-cell subsets has been found in subjects who harbored X4/SI variants,17,18 which may be related to the almost exclusive expression of CXCR4 coreceptor on this subset, while CCR5 predominates on memory and activated T cells.19 CXCR4 is also highly expressed on several subsets of thymocytes, and the thymus microenvironment has been shown to favor the replication of X4 variants.20,45 Moreover, increased plasma levels of interleukin-7 induced by CD4+ T-cell depletion upregulate CXCR4 expression on CD4+ T cells and thymocytes and may promote the emergence of X4 variants.45,46 Additionally, X4 variants were found to have higher replication capacity on HAART compared with their R5 counterparts.47 Thus, the preferential replication of CXCR4-using viruses in the microenvironment of residual virus production and their selection in thymocytes or naive CD4+ T cells is compatible with the emergence of X4 variants in cellular reservoirs during immune reconstitution on HAART.
 
However, an alternative mechanism of the R5 to X4 switch that we observed during HAART could only be a passive one, through the peripheral expansion of long-lived cells harboring pre-HAART archival X4 variants. The long-lived population of T cells consists of primarily naive CD45RA+ T cells, while the majority of CD45RO+ memory T cells have relatively shorter life spans.48 Thus, the emergence of X4 variants in cellular reservoirs might result from a differential turnover between cells that mainly harbor X4 variants, such as long-lived naive CD4+ T cells, and cells that mainly harbor R5 variants, such as CCR5+ CD4+ memory T cells, the majority of which are destined to die rapidly after proliferation and elaboration of effector function.48
 
In our study, clonal analysis of HIV-1 quasipecies performed in patients for whom an R5 to X4 switch occurred in the majority virus population during HAART (R5/X4 group) revealed that minority virus populations of X4 variants were usually present within the virus quasispecies at baseline. Phylogenetic analysis revealed that the emergent X4 variants on HAART were related to these minority species present in the plasma or PBMCs at baseline. The presence of minority virus populations of pathogenic X4 variants might also explain that baseline CD4+ T-cell counts were as low in the R5/X4 group as they were in the X4/X4 group. These findings suggest that HAART may produce the conditions necessary for the gradual selection of preexisting minor X4 variants in cellular reservoirs but do not prefigure whether this selection occurs passively through the progressive selection of latently X4-infected long-lived cells or actively through the preferential residual replication of X4 variants during HAART.
 
Whether the emergence of a majority virus population of X4 variants in cellular reservoirs that we observed may have a clinical impact in patients with sustained suppression of plasma viremia remains to be determined. However, most of the patients we studied who had a majority virus population of X4 variants, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously R5 variants throughout follow-up. In agreement with our findings, it has recently been established in a larger group of HIV-1-infected individuals who initiated HAART that the X4 genotype based on the 11/25 rule in baseline plasma is an independent predictor of poor immunologic response and more rapid mortality, even in individuals who achieved plasma virus suppression to <500 HIV-1 RNA copies/mL.49 A study also suggested that children infected with SI variants of HIV-1 had lower thymic output than those infected with NSI variants.50 In our study, the impaired immune reconstitution in patients harboring X4 variants might be related to pre-HAART damages induced by X4 variants on the naive CD4+ T-cell pool and the thymus or to the consequences of the residual replication of highly pathogenic X4 variants on HAART.
 
In conclusion, we have shown for the first time that an R5 to X4 switch in the majority virus population can occur despite effective HAART, suggesting that potent antiretroviral therapy produces the conditions necessary for the gradual emergence of X4 variants in cellular reservoirs. Impaired CD4+ T-cell restoration in these patients suggests that this emergence of X4 variants in patients on HAART is clinically relevant. Additional studies of virus genetic evolution in patients on HAART are needed to better understand the mechanisms and the pathogenic consequences of change in coreceptor usage during potent antiretroviral therapy.
 
INTRODUCTION
The clinical course of HIV-1 infection is highly variable between patients. Among the viral factors that may affect the rate of disease progression, HIV-1 coreceptor usage plays a critical role for virus tropism and pathogenesis. The chemokine receptors CCR5 and CXCR4 are the principal coreceptors for entry of HIV-1 into target cells (for a review, see Berger et al1). CCR5-using viruses (R5 variants) predominate in the initial stages of infection, suggesting that they are selected during or shortly after transmission. A switch from CCR5 to CXCR4-using viruses (X4 variants), initially identified as a switch from non-syncytium-inducing (NSI) to syncytium-inducing (SI) phenotype,2 occurs later in the natural course of infection in about half of HIV-infected individuals and correlates with the subsequent accelerated decrease in CD4+ T-cell count and disease progression.3-6
 
Potent combination antiretroviral therapy (highly active antiretroviral therapy or HAART) is highly efficient at reducing virus replication and increasing CD4+ T-cell numbers, resulting in significant decreases in AIDS-associated morbidity and mortality. In response to HAART, HIV-1 RNA levels in the plasma often decline to below the limit of detection by sensitive molecular assays in many patients. However, cells harboring replication-competent HIV-1 persist even in patients who have been on suppressive HAART for many years, which is a major obstacle to virus eradication.7 Resting memory CD4+ T cells harboring latent HIV-1 are the most significant cellular reservoir of virus. This pool of long-lived latently infected CD4+ T cells is established early during primary HIV-1 infection and has minimal decay despite prolonged suppressive HAART, enabling the prolonged persistence of HIV-1.8,9 Moreover, low levels of ongoing virus replication can be detected in most patients whose plasma viremia is apparently suppressed on HAART,10-13 and it has been suggested that this residual virus replication may replenish the latent reservoir and contribute to its apparent stability.14
 
Significant virus genetic evolution, especially in the HIV-1 envelope gene (env), has been observed in some patients on effective HAART, but whether this evolution might lead to changes in coreceptor usage of HIV-1 during HAART is not known.15-17 An R5 to X4 switch of HIV-1 variants on HAART could have important implications as the target cell population of CXCR4-using viruses is significantly expanded to include naive CD4+ T cells and thymocytes,18-21 which are known to play a critical role in immune reconstitution on HAART.22-23
 
The present longitudinal study of 32 patients with prolonged suppression of plasma viremia on HAART for 5 years analyzes the evolution of the 3rd variable region (V3) of env and its impact on coreceptor usage and CD4+ T-cell restoration.
 
RESULTS
 
Patient Characteristics

The subjects were 26 men and 6 women, with a mean age of 38.3 years (range, 25-54). The median baseline CD4+ T-cell count was 40 cells/mm3 (IQR, 21-116), and the median baseline plasma log10 HIV-1 RNA was 5.0 copies/mL (IQR, 4.5-5.7). The median duration of HIV-1 infection before the initiation of HAART was 96 months (IQR, 60-132). All 32 patients showed a rapid decline in plasma HIV-1 RNA after the initiation of HAART and had undetectable HIV-1 RNA in the plasma after 6 months. The plasma HIV-1 RNA in all the patients remained undetectable by the ultrasensitive RT-PCR assay throughout 5-year follow-up (except for patients 3 and 4, who were lost of follow-up after month 30). There was a gradual increase in the CD4+ T-cell count of all the patients in response to HAART. The median increase in CD4+ T-cell count was +81 cells/mm3 (IQR, 41-123) after 6 months of treatment, +114 cells/mm3 (IQR, 86-170) after 12 months, +264 cells/mm3 (IQR, 182-354) after 30 months, and +350 cells/mm3 (IQR, 263-541) after 60 months of HAART. Phylogenetic analysis of the env fragment showed that all strains belonged to subtype B, except one (patient 2) that belonged to recombinant subtype CRF01-A/E (data not shown).
 
Changes in the Predicted Coreceptor Usage of the Predominant Virus Population During HAART
The predicted coreceptor usage of the majority virus population over the sequential samples from each patient was determined from the V3 env region amino acid sequence. Uncharged residues at position 11 of V3 (mostly serine/glycine), negatively charged residues at position 25 (mostly glutamic/aspartic acid), and a net charge of the V3 loop <+5 have been reported to predict CCR5 chemokine receptor usage.24,25 Conversely, positively charged residues at position 11 or 25 (mostly arginine/lysine) and a net charge of the V3 loop ³+5 have been reported to predict CXCR4 chemokine receptor usage.26-28 Loss of an N-linked glycosylation site within the V3 region has also been reported to have a major influence on the virus switching from the R5 to X4 phenotype.30,31 The presence of a positively charged residue at position 11 or 25 was used in our study as the primary criterion for predicting the X4 phenotype from the virus genotype. Most of the sequences identified as X4 variants had a net charge of the V3 loop ³+5 concordant with the 11/25 rule for coreceptor usage prediction. The virus in 3 patients (patients 2, 3, and 8) that had sequences without positively charged residue at positions 11 or 25 but had lost the N-linked glycosylation site, or had a high net positive charge of the V3 loop, were classified as X4 variants. We also used a recently described position-specific scoring matrix that reliably predicts X4/SI phenotype in patients harboring subtype B viruses (all patients except patient 2),29 and we found concordant predictions of coreceptor usage (data not shown).
 
Baseline V3 env sequences were analyzed from HIV-1 RNA in the plasma for all patients and from HIV-1 DNA in total PBMCs for 7 patients before the initiation of HAART; subsequent analyses were then only performed from HIV-1 DNA in total PBMCs since all patients had sustained suppression of plasma HIV-1 RNA on HAART. The plasma of 23 of the 32 patients harbored a majority virus population of R5 variants at baseline before starting HAART. Nine patients had a majority virus population of X4 variants at baseline. In 7 patients (patients 2, 6, 9, 18, 20, 24, and 25), both baseline plasma and PBMC samples were available for analysis; it showed that the majority virus populations found in the plasma and PBMCs at baseline were almost identical. Virus tropism was next determined after about 30 and 60 months of suppressive HAART by sequencing HIV-1 DNA from PBMCs. R5 variants remained predominant at subsequent times in 12 of the 23 patients who harbored a majority virus population of R5 variants at baseline (R5/R5 group). In contrast, the remaining 11 patients showed a progressive emergence of X4 variants (R5/X4 group). In 9 of these patients, the R5 to X4 switch in the majority virus population occurred before month 30. We did not observe any reversion from X4 to R5 variants at month 60 analysis. In the 2 remaining patients (patients 7 and 12), the R5 to X4 switch occurred between months 30 and 60 of HAART. Finally, in patients who harbored a majority virus population of X4 variants at baseline, X4 variants remained predominant at subsequent times in 8 of 9 patients, and in 1 patient (patient 26), R5 variants were transiently predominant at mont
 
h 30 but X4 variants were again predominant at month 60 (X4/X4 group). Detailed Genotypic Characterization and Evolution of Virus Populations in Patients Harboring X4 Variants on HAART
We performed clonal analysis in 8 patients harboring X4 variants on HAART to take into account both the majority and minority virus populations. The sequences obtained by bulk sequencing precisely matched consensus sequences obtained by clonal analysis at nearly all codons of the env fragment and provided concordant prediction of coreceptor usage. Thus, direct sequencing of the V3 env region accurately reflects the majority virus population. HIV-1 quasispecies were characterized in baseline samples of 5 patients (patients 4, 6, 7, 17, and 20) for whom an R5 to X4 switch in the majority virus population occurred on HAART (R5/X4 group). We found minority populations of X4 variants in baseline plasma or PBMCs of 3 of these 5 patients (patients 4, 6, and 20). Clonal analyses of virus populations are shown for 2 patients (patients 6 and 20) for whom baseline PBMC samples were available. These patients harbored minor X4 variants in baseline plasma or PBMCs that were identical or closely related to those viruses that subsequently became predominant in PBMCs during HAART. Phylogenetic trees were constructed using the neighbor-joining method for the env sequences found in PBMCs at baseline and at month 30 in patients 6 and 20; both revealed that the predominant population of X4 variants found at month 30 was derived from the initially minority population of X4 variants present at baseline.
 
A detailed characterization of virus populations was also performed in baseline PBMCs for 3 patients (patients 2, 9, and 18) who continuously harbored predominantly X4 variants from baseline (X4/X4 group). In patients 2 and 18, the majority of the virus population at baseline consisted of X4 variants, although minor R5 variants were also found in PBMCs. However, these minority populations of R5 variants were not selected during HAART, and the X4 variants remained largely predominant in PBMCs on HAART in these patients. In patient 9, only X4 variants were found in baseline PBMCs, and these viruses remained predominant in subsequent analysis performed on HAART.
 
Impact of Coreceptor Usage of the Majority Virus Population on CD4+ T-Cell Count on HAART
We next determined whether the presence of X4 variants in cellular reservoirs influenced the restoration of CD4+ T cells in response to suppressive HAART. Patients in whom a switch in the majority virus population from R5 to X4 variants was detected (R5/X4 group, n = 11) were compared with patients who continuously harbored predominant R5 variants (R5/R5 group, n = 12) and with patients who continuously harbored predominant X4 variants (X4/X4 group, n = 9) throughout follow-up. The 3 groups were comparable regarding the mean age (R5/R5 group, 39.3 years; R5/X4 group, 36.6 years; X4/X4 group, 39 years; P = 0.47); the median duration of HIV infection before HAART (R5/R5 group, 90 months; R5/X4 group, 84 months; X4/X4 group, 96 months; P = 0.92); and the median baseline plasma HIV-1 RNA load (R5/R5 group, 4.95 log10 copies/mL; R5/X4 group, 5 log10 copies/mL; X4/X4 group, 5 log10 copies/mL; P = 0.88). The median baseline CD4+ T-cell count was lower in the X4/X4 group (39 cells/mm3) and in the R5/X4 group (25 cells/mm3) than in the R5/R5 group (110 cells/mm3), but this difference was not statistically significant (P = 0.29). The CD4+ T-cell counts during HAART in the 3 groups of patients are shown in Figure 5. The patients harboring a majority virus population of X4 variants during HAART, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously a majority virus population of R5 variants. The median increase in CD4+ T cells from baseline was +301 cells/mm3 (IQR, 189-459) after 60 months of HAART in patients harboring a majority virus population of X4 variants on HAART and +442 cells/mm3 (IQR, 306-650) in patients harboring only R5 variants (P = 0.07).
 
METHODS
 
Patients

Subjects (n = 32) were randomly selected from a well-characterized cohort of HIV-1-infected patients treated at the Department of Infectious Diseases of Toulouse University Hospital (Toulouse, France) based on sustained suppression of plasma HIV-1 RNA on HAART for about 5 years, as assessed at 3-month intervals, and cryopreserved samples' availability. All patients had been placed on a first triple-drug regimen consisting of 2 nucleoside analogue reverse transcriptase inhibitors (NRTIs) (zidovudine or stavudine plus lamivudine) and 1 protease inhibitor (indinavir or ritonavir). Most of them had previously experienced multiple failures of NRTI-based therapies. Informed consent was obtained for all the subjects.
 
Plasma HIV-1 RNA
Plasma HIV-1 RNA was assessed at 3-month intervals using the Amplicor HIV-1 Monitor reverse transcriptase polymerase chain reaction (RT-PCR) assay (Roche Diagnostics, Meylan, France), with a lower limit of detection of 200 copies/mL during the first 2 years of the study, and then with the ultrasensitive Amplicor HIV-1 Monitor RT-PCR assay (Roche Diagnostics), with a lower limit of detection of 20 copies/mL, during the next 3 years.
 
CD4+ T-Lymphocyte Count
Peripheral blood CD4+ T lymphocytes were quantified at 3-month intervals by flow cytometry (Epics Profile, Beckman-Coulter, Villepinte, France) using commercially available monoclonal antibodies (Becton-Dickinson, Le Pont de Claix, France).
 
Sequencing of the V3 Region of HIV-1 env
A sequence spanning the HIV-1 V3 env region was amplified from frozen plasma taken at baseline before the initiation of HAART. Baseline V3 env sequences were also amplified from total peripheral blood mononuclear cells (PBMCs) from 7 patients for whom cryopreserved samples were available; subsequent analyses were then only performed from HIV-1 DNA in PBMCs since all patients had sustained suppression of plasma HIV-1 RNA throughout follow-up. V3 env sequences were amplified from total PBMCs after about 30 and 60 months of effective HAART. Nucleic acid isolation, cDNA synthesis, and PCR amplifications were performed as previously described.17 Briefly, a nested PCR was used to amplify a region of 667 nucleotides spanning the V3 env region using E1 (sense primer, 5Aa-TTAGGCATCTCCTATGGCAGGAAGCGG-3Aa; nucleotides 5956-5985 of the HIVHXB2 genome) and E2 (antisense primer, 5Aa-AGTGCTTCCTGCTGCTCCCAAGAACCCAAG-3Aa; 7810-7781) as outer primers, and E3 (sense primer, 5Aa-CTGTTAAATGGCAGTCTAGC-3Aa; 7001-7020) and E4 (antisense primer, 5Aa-CACTTCTCCAATTGTCCCTCA-3Aa; 7661-7647) as inner primers. Endpoint dilution PCR amplifications from PBMCs of patients of the cohort who have sustained undetectable plasma virus loads suggest that at least 50-100 target molecules were subjected to PCR amplification in each experiment.17 The PCR products from 3 separate positive amplifications were pooled before being sequenced in the sense and antisense directions by the dideoxy chain-termination method (Big Dye Terminators v.3.1, Applied Biosystems, Paris, France) on an ABI 3100 DNA sequencer (Applied Biosystems). Bulk sequencing enabled us to determine the V3 env genotype of the majority virus population. In contrast to the V1 and V2 env regions, length polymorphisms of the V3 env region were rarely found in the virus population of a given individual and did therefore not represent an obstacle for bulk sequencing of the V3 env region.
 
Cloning of PCR Products
Clonal analysis of the PCR products was performed for 8 patients to take into account both the majority and minority virus populations. The PCR products were cloned using the TA cloning kit (Invitrogen, Cergy Pontoise, France). Plasmid DNAs containing V3 env inserts were purified using QIAprep 8 miniprep kit (Qiagen), and multiple molecular clones (mean, 20; range, 15-23) were sequenced in both directions.
 
Analysis of Sequence Data
Electrophoretogram data were analyzed using the Sequence Navigator program (Applied Biosystems). Multiple alignments were done with the CLUSTALW 1.7 ( http://www.ebi.ac.uk ) program. Dendograms were created by the neighbor-joining method with the Phylogeny Inference Package (PHYLIP ( http://evolution.genetics.washington.edu/phylip.html )) and tree diagrams were plotted with the TREEVIEW 1.66 ( http://taxonomy.zoology.gla.ac.uk/rod/rod.html ) program. Bootstrap analysis consisting of 100 replicates was performed on the neighbor-joining trees. The frequency with which the node occurred is indicated at each relevant branch point.
 
Determination of the Virus R5/X4 Genotype
The predicted coreceptor usage of HIV-1 was determined from the V3 env region amino acid sequence. R5 and X4 variants were identified according to the critical amino acid residues at positions 11 and 25 and the net charge of the V3 region (calculated by subtracting the number of negatively charged amino acids D and E from the number of positively charged ones K and R) and were confirmed by using a position-specific scoring matrix.24-29 It should be noted that genotypic approaches to determine HIV-1 coreceptor usage imply that some of the sequences amplified might correspond to defective viruses.
 
Statistical Analysis
Quantitative data were summarized using median and interquartile range (IQR). Comparisons between groups were performed by using the nonparametric Kruskall-Wallis H test. A value of P < 0.05 was considered statistically significant. Nucleotide sequence accession numbers: The sequences reported here were given GenBank accession numbers AY530634-AY530727.
 
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