HIV-1 subtype C envelope characteristics associated with divergent rates of chronic disease progression

Abstract

Background: HIV-1 envelope diversity remains a significant challenge for the development of an efficacious vaccine. The evolutionary forces that shape the diversity of envelope are incompletely understood. HIV-1 subtype C envelope in particular shows significant differences and unique characteristics compared to its subtype B counterpart. Here we applied the single genome sequencing strategy of plasma derived virus from a cohort of therapy naïve chronically infected individuals in order to study diversity, divergence patterns and envelope characteristics across the entire HIV-1 subtype C gp160 in 4 slow progressors and 4 progressors over an average of 19.5 months.

Results: Sequence analysis indicated that intra-patient nucleotide diversity within the entire envelope was higher in slow progressors, but did not reach statistical significance (p = 0.07). However, intra-patient nucleotide diversity was significantly higher in slow progressors compared to progressors in the C2 (p = 0.0006), V3 (p = 0.01) and C3 (p = 0.005) regions. Increased amino acid length and fewer potential N-linked glycosylation sites (PNGs) were observed in the V1-V4 in slow progressors compared to progressors (p = 0.009 and p = 0.02 respectively). Similarly, gp41 in the progressors was significantly longer and had fewer PNGs compared to slow progressors (p = 0.02 and p = 0.02 respectively). Positive selection hotspots mapped mainly to V1, C3, V4, C4 and gp41 in slow progressors, whereas hotspots mapped mainly to gp41 in progressors. Signature consensus sequence differences between the groups occurred mainly in gp41.

Conclusions: These data suggest that separate regions of envelope are under differential selective forces, and that envelope evolution differs based on disease course. Differences between slow progressors and progressors may reflect differences in immunological pressure and immune evasion mechanisms. These data also indicate that the pattern of envelope evolution is an important correlate of disease progression in chronic HIV-1 subtype C infection.

Figure 1

CD4 of study entry, study exit and latest available time-point data for slow progressors and progressors. The red circles depict the data points for the slow-progressors. The blue squares depict data points for the progressors. Red bars and blue bars represent the p values for the slow progressors and progressors respectively. Black bars represent p values for inter-group comparison for the different time-points. NS = not significant. All comparisons between the study entry, study exit and latest available time-point parameters were performed using the Mann-Whitney unpaired t test, and p values are shown. Differences were regarded as statistically significant with a p value < 0.05. When slow progressors were compared to progressors, the analysis yielded significant differences when the CD4 at study exit and last available time-points were compared - as shown above (p = 0.04 and p = 0.02 respectively). Likewise viral load was significantly different between the groups at study exit and the latest available time-point (p = 0.03 and p = 0.02 respectively, data not shown).

Figure 2

Maximum Likelihood trees of SGA-derived full-length env sequences from Progressors and Slow progressors. Figure 2A Subtype tree of consensus sequences for slow progressors entry (●) and exit (○) and progressors entry (■) and exit (□) time-points. Subtype reference strains were obtained from the Los Alamos database (http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html). The tree was rooted with Group O as the outgroup. Figures 2B to 2E represent maximum likelihood trees for the slow progressor sequences and Figures 2F to 2I represent trees for the progressor sequences. All trees were drawn in Paup* using the appropriate substitution model. Bootstrap support from 1000 bootstrap resamplings is indicated by ●. Only values >70% are shown. The scale bar is shown at the bottom of figure 2A is 0.1 and for figures 2B-2I the scale bar is 0.005. The mean study entry and exit intra-patient nucleotide diversity and the standard error of (SE) for both the groups are shown in the tables below the individual trees.

Figure 3

Box-and-whisker plots of genetic diversity of the dissected envelope gene for V1, V2, C2, V3, C3, V4, C4 and V5 and gp41 for slow progressors and progressors. The whiskers extend to the upper and lower adjacent values. Comparisons between the groups were done with the Mann Whitney unpaired t test, and p values are shown. Correlations were regarded as statistically significant with a p value < 0.05 and only significant p values are shown. p values depicted with an asterisk (*) indicate the ones corrected for multiple comparisons using the Bonferroni correction of p ≤ 0.006. Mean diversity value is depicted as (+). Figure 3A Diversity of V1, V2, C2, V3, C3, V4, C4, V5 and gp41 in slow progressors (SP) and progressors (P) overall. Figure 3B Box and whisker plots of intra-patient diversity analysis for slow progressors for different regions of the Env gene for study entry and study exit. Figure 3C Box and whisker plots of intra-patient diversity analysis for progressors for different regions of the Env gene for study entry and study exit.

Figure 4

Three dimensional structural illustrations of positions associated with positive negative and neutral selection. Locations were mapped onto a model of gp120 based on the X-ray structure of the gp120 core in complex with sCD4 and 21c Fab (3LQA.pdb) for slow progressors - Figure 4A and for progressors - Figure 4C. V1V2 and V3 loops were drawn onto the core for completeness. In the orientation shown, the cellular and viral membranes would be located above and below the protein respectively. Figure 4B and 4D represent ribbon structures of gp41 for slow progressors and progressors with the MPER region highlighted. Cartoon diagrams showing locations under positive selection, as determined by dN/dS ratios for subtype C sequences. Red indicates strong positive selection (dN/dS >4) as shown above in HXB2 positions 87, 336, 340, 396, 410 and 460 for slow progressors (Figure 4A) and in progressors at positions 350 (Figure 4C) and 607, 612 and 641 in Figure 4D. Blue indicates strongly negatively selected positions (<-3). Purple and purple arrows denote changes in putative functional sites as shown in Figures 4B, 4C and 4D. Spheres indicate signature sequence differences. It should be noted that the gp120 core crystal structures which were modeled on the 3LQA.PDB structure, include amino acid residues from HXB2 position 86-491. The gp41 structure based on 1ENV.pdb includes amino acid residues from HXB2 position 541-662. Therefore all the positively and negatively selected sites are not indicated on the gp120 and gp41 structures.