Intercompartmental recombination of HIV-1 contributes to env intrahost diversity and modulates viral tropism and sensitivity to entry inhibitors

Abstract

HIV-1 circulates within an infected host as a genetically heterogeneous viral population. Viral intrahost diversity is shaped by substitutional evolution and recombination. Although many studies have speculated that recombination could have a significant impact on viral phenotype, this has never been definitively demonstrated. We report here phylogenetic and subsequent phenotypic analyses of envelope genes obtained from HIV-1 populations present in different anatomical compartments. Assessment of env compartmentalization from immunologically discrete tissues was assessed utilizing a single genome amplification approach, minimizing in vitro-generated artifacts. Genetic compartmentalization of variants was frequently observed. In addition, multiple incidences of intercompartment recombination, presumably facilitated by low-level migration of virus or infected cells between different anatomic sites and coinfection of susceptible cells by genetically divergent strains, were identified. These analyses demonstrate that intercompartment recombination is a fundamental evolutionary mechanism that helps to shape HIV-1 env intrahost diversity in natural infection. Analysis of the phenotypic consequences of these recombination events showed that genetic compartmentalization often correlates with phenotypic compartmentalization and that intercompartment recombination results in phenotype modulation. This represents definitive proof that recombination can generate novel combinations of phenotypic traits which differ subtly from those of parental strains, an important phenomenon that may have an impact on antiviral therapy and contribute to HIV-1 persistence in vivo.

Fig. 1.

Phylogenetic assessment of env compartmentalization from immunologically discrete tissues. A midpoint-rooted ML phylogenetic reconstruction for the combined HIV-1 env alignment generated using the GTR+I+Γ₄ substitution model (74, 88, 89) is shown. Branch lengths are in accordance with the scale bar and are proportional to genetic distance. Significance of clustering of isolates was assessed via bootstrap resampling of the sequence data derived from 1000 replications. *, Values >70%; **, values >99%. Isolates marked with a “+” were selected for subsequent phenotyping.

Fig. 2.

Evidence for intercompartment mosaicism. Example similarity plots and informative site arrays were used to characterize intercompartment mosaics. Plots were generated in SimPlot with a sliding window size of 200 bp and a step size of 20 bp and represent a query mosaic compared to two parental sequences. Dotted vertical lines indicate absolute breakpoint positions correlated with diversity plot crossover points. Associated P values (*, P < 0.05; **, P < 0.01; ***, P < 0.001) are derived from informative site arrays (described in Materials and Methods and displayed in Table 2). Relative numbers of informative sites shared by query sequences with parental strains are displayed below regions of differential homology. Four taxon trees consistent with these sites are displayed to the left: QS, query sequence; PS1, parental sequence 1; PS2, parental sequence 2; OG, outgroup. (A) Query mosaic E21BrD107 compared to parental sequences E21BrD90 and E21LnD56. (B) Query mosaic KM11BlR7 compared to parental sequences KM11BlR22 and KM11SeR2. (C) Query mosaic KM34SeR63 compared to parental sequences KM34BlR11 and KM34SeR26. (D) Schematic diagram depicting breakpoint locations in eight characterized mosaics, displayed relative to the positions of functionally defined regions in the HXB2 reference env located above. The patient from which each mosaic was isolated is detailed to the right of each individual recombinant env schematic. Regions of differential homology are color coded.

Fig. 3.

Phylogenetic trees corresponding to regions of differential homology in intercompartment mosaic E21BrD107. Patient E21's env alignment was divided into four segments, in agreement with the three breakpoints identified in isolate E21BrD107, and ML phylogenetic trees were constructed for each segment: positions 1 to 262 (A), positions 263 to 774 (B), positions 775 to 1514 (C), and positions 1515 to 2571 (D). Highlighted isolate E21BrD107 exhibits significant positional shifting between compartment specific clades.

Fig. 4.

Macrophage tropism of HIV-1 gp160s amplified from anatomically discrete tissues. Patient/tissue-specific gp160s were expressed on HIV-1 pseudovirions. env⁺ pseudovirions were titrated on HeLa TZM-BL cells and on primary macrophages. Macrophage infectivity is expressed as a percentage of HeLa TZM-BL cell infectivity.

Fig. 5.

Amino acid sequence alignments for functional gp160s. Functional gp160s from patient E21 (A) and patient KM34 (B) are aligned relative to the HXB2 reference strain, and all residue numbering is relative to homologous positions in HXB2 gp160. Within the alignment, asparagine (N) residues that are likely to be glycosylated have been replaced by “#”. Horizontal lines located above alignment segments indicate the position of the variable regions V1 to V5. Symbols or letters located directly above alignment segments highlight the functionally important residues: *, CD4 contact residue; m, additional residue previously implicated in modulation of macrophage tropism; b, residue previously implicated in modulating sensitivity to b12; and g, residue previously implicated in modulating sensitivity to 2G12.

Fig. 5.

Amino acid sequence alignments for functional gp160s. Functional gp160s from patient E21 (A) and patient KM34 (B) are aligned relative to the HXB2 reference strain, and all residue numbering is relative to homologous positions in HXB2 gp160. Within the alignment, asparagine (N) residues that are likely to be glycosylated have been replaced by “#”. Horizontal lines located above alignment segments indicate the position of the variable regions V1 to V5. Symbols or letters located directly above alignment segments highlight the functionally important residues: *, CD4 contact residue; m, additional residue previously implicated in modulation of macrophage tropism; b, residue previously implicated in modulating sensitivity to b12; and g, residue previously implicated in modulating sensitivity to 2G12.

Fig. 6.

Sensitivity of HIV-1 gp160s to maraviroc. Patient/tissue-specific env⁺ pseudovirions were tested for sensitivity to entry inhibition by maraviroc.