High-Sequence Diversity and Rapid Virus Turnover Contribute to Higher Rates of Coreceptor Switching in Treatment-Experienced Subjects with HIV-1 Viremia

Abstract

Coreceptor switching from CCR5 to CXCR4 is common during chronic HIV-1 infection, but is even more common in individuals who have failed antiretroviral therapy (ART). Prior studies have suggested rapid mutation and/or recombination of HIV-1 envelope (env) genes during coreceptor switching. We compared the functional and genotypic changes in env of viruses from viremic subjects who had failed ART just before and after coreceptor switching and compared those to viruses from matched subjects without coreceptor switching. Analysis of multiple unique functional env clones from each subject revealed extensive diversity at both sample time points and rapid diversification of sequences during the 4-month interval in viruses from both 9 subjects with coreceptor switching and 15 control subjects. Only two subjects had envs with evidence of recombination. Three findings distinguished env clones from subjects with coreceptor switching from controls: (1) lower entry efficiency via CCR5; (2) longer V1/V2 regions; and (3), lower nadir CD4 T cell counts during prior years of infection. Most of these subjects harbored virus with lower replicative capacity associated with protease (PR) and/or reverse transcriptase inhibitor resistance mutations, and the extensive diversification tended to lead either to improved entry efficiency via CCR5 or the gain of entry function via CXCR4. These results suggest that R5X4 or X4 variants emerge from a diverse, low-fitness landscape shaped by chronic infection, multiple ART resistance mutations, the availability of target cells, and reduced entry efficiency via CCR5.

Keywords: HIV-1 coreceptor switching; sequence diversity; sequence evolution.

FIG. 1.

(A) Distribution of entry phenotypes by coreceptor use for all functional HIV envelope (env) clones with unique sequences. Subjects with R5X4 or X4 env clones still had a predominance of R5 env clones (e.g., see Fig. 2 and Supplementary Figs. S1–S24). (B) Interval between sample collections for subjects who maintained R5 use, who switched from R5 to R5X4 (or X4), or who maintained R5X4 use.

FIG. 2.

(A) Entry data for HIV env clones from one subject before coreceptor switching (S5) and 151 days later (S6) when enhanced Trofile assay results revealed DM viral variants. Data are entry of pseudotyped env clones into target cells expressing either CCR5 (x-axis) or CXCR4 (y-axis) with a reporter construct expressing luciferase. Data are plotted as log₁₀ RLU with significant entry function being greater than 10³ RLU (indicated by gray line on each axis). (B) Amino acid pairwise distance of regions of gp120 from all S5 sequences to the unique sequence of R5X4 env clone S6.58 (see A). (C) Phylogenetic tree of full-length/gp160 env sequences from time point S5 and S6. Scale is 0.01% or 1% per 100 amino acids. Note that R5X4 env clone S6.58 is the most divergent sequence identified. Mean pairwise distance per 100 amino acids is given at the bottom of (C). A similar format is used for Supplementary Figures 1–24. DM, dual/mixed; RLU, relative light units.

FIG. 3.

(A) Mean amino acid divergence/100 amino acids (AA) for all subjects who maintained CCR5 use at both sample time points (R5 to R5), all subjects who had some R5X4 or X4 env clones at the second sample time point (R5 to R5X4), two subjects who had R5X4 env clones at multiple time points (R5X4 to R5X4). All data points are displayed in a box and whisker plot with the box representing the upper and lower quartile, the line in the box representing the median value, and the whiskers the data range. (B) Mean pairwise distance/100 amino acids for the same data. One subject with extreme divergence was excluded from these data because of suspected superinfection or dual infection (Fig. 9). AA, amino acids.

FIG. 4.

(A) The same data as presented in Figure 3 but plotted as divergence/100 AA/year to correct for differences in the duration between the two sample time points. (B) Data from Figure 3 plotted as mean pairwise distance/100 AA/year to correct for differences in the duration between the two sample time points (Table 1).

FIG. 5.

(A) Percent divergence in amino acid sequence of gp120 (535–560 amino acids) from the consensus of all R5 env clones at the first sample time point to the R5X4 env clone that mediated the most efficient entry via CXCR4 at the second time point (e.g., clone 6.58 in Fig. 2). (B) Changes in V3 sequences between the first and second sample point for R5 to R5 controls and R5 to R5X4 coreceptor switch variants.

FIG. 6.

(A) Mean entry via CCR5 in log₁₀ RLU for all env clones from subjects who maintained CCR5 or CXCR4 use (NS) or subjects who switched to R5X4 or X4 (S). p value of paired, two-tailed T-test is shown, which indicates significantly lower entry function via CCR5 for env clones from subjects with coreceptor switching. (B) The same data, but plotted for sample time point 1 (first) and sample time point 2 (second) for both NS and S subjects. NS, nonswitch; S, switch. NS, non-switch controls when column label; ns, non-significant when referring to statistical test shown in Figure panel

FIG. 7.

(A) Average V1/V2 length for env sequences from subjects who maintained R5 viruses at both sample time points compared to subjects who switched from R5 to R5X4 or X4 viruses at the second sample time point. p value from paired, two-tailed T-test. (B) Change in V1/V2 length between the first and second sample time point from the same env sequences presented in (A). The trend toward a shorter V1/V2 region following coreceptor switching was not significant by the Mann–Whitney nonparametric test.

FIG. 8.

(A) Nadir CD4 T cell counts for subjects with R5 viruses at both sample time points compared with those viruses that switched from R5 to R5X4 or maintained R5X4 tropism. p value calculated from paired, two-tailed T-test. (B) Months since last nadir viral load measurement for the same subjects. T-test indicated that the difference was NS. NS, nonsignificant.

FIG. 9.

Neighbor-joining phylogenetic tree from dual/superinfected subject. The tree includes 30 env sequences from subject 3, and 102 and 290 randomly chosen subtype B sequences (see Materials and Methods section). Major and minor clades found in this individual are shown in yellow and red highlighted regions, respectively. Two recombinant env sequences containing regions derived from both the major and minor clades are in red type and indicated with arrows.