Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression

Abstract

There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates (r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases (P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants (P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes.IMPORTANCE HIV-1 subtypes are unevenly distributed globally, and there are reported differences in their rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that HIV-1 Gag-protease-driven replication capacity correlates with the replication capacity of whole virus isolates. We further show that subtype B displays a significantly higher Gag-protease-mediated replication capacity than does subtype C, and we identify a major genetic determinant of these differences. Moreover, in two independent East African cohorts we demonstrate a reproducible hierarchy of Gag-protease-driven replicative capacity, whereby recombinants exhibit the greatest replication, followed by subtype D, followed by subtypes A and C. Our data identify Gag-protease as a major determinant of subtype differences in disease progression among HIV-1 subtypes; furthermore, we propose that the poorer viral replicative capacity of subtypes A and C may paradoxically contribute to their more efficient spread in sub-Saharan Africa.

Keywords: Gag-protease; HIV-1 subtype; viral replication capacity.

FIG 1

Contribution of Gag-protease to overall HIV-1 replication capacity. The graph shows a significant positive correlation (Pearson's correlation) between the replication capacities (RC) of HIV-1 subtype C isolated from patients and the corresponding NL4-3 recombinant viruses encoding Gag-protease derived from the same patients. Replication capacity was assayed in GXR cells using flow cytometry and normalized to the growth of the wild-type NL4-3 virus (replication capacity of 100%).

FIG 2

HIV-1 subtype B versus subtype C Gag-protease-mediated replication capacity. (A) Significant differences between the replication capacities of NL4-3 recombinant viruses encoding subtype B (n = 803) (41) and subtype C (n = 406) (39) Gag-proteases from patients chronically infected with HIV-1. (B) A comparison of subtype B (n = 25) and subtype C (n = 406) Gag-protease-mediated replication capacities performed in the same laboratory. (C) Significant differences between subtype B (n = 25) and subtype C (n = 25) Gag-protease-mediated replication capacity remain when samples matched for viral loads and CD4 counts are compared. Replication capacity was assayed in GXR cells using flow cytometry and normalized to the growth of the wild-type NL4-3 virus (replication capacity of 100%). The Mann-Whitney U test was used to test for significance, and lines represent the means.

FIG 3

Replication kinetics of the subtype C consensus Gag pZM246-F10 and NL4-3 Gag pZM246-F10 recombinant viruses. Comparison of the replication kinetics of pZM246-F10 (subtype C) viral constructs carrying the gag genes from subtype C consensus and NL4-3. The wild-type pZM246-F10 virus was included as a control. The replication assay was carried out in activated pooled PBMCs from four donors and monitored using p24 ELISA.

FIG 4

Replication kinetics of consensus C Gag p24 NL4-3 recombinant virus and NL4-3 wild-type virus. The replication of NL4-3 encoding consensus C Gag p24 is similar to that of wild-type NL4-3. The replication assay was carried out in GXR cells, and viral replication was monitored using flow cytometry to measure GFP expression.

FIG 5

Replication capacities of HIV-1 subtype B and C viruses encoding subtype-specific mutations in Gag. (A) Replication capacities of subtype B NL4-3 wild-type virus and NL4-3 viruses encoding subtype C-specific Gag residues 67S, 473A, and 483−/484−. (B) Replication capacities of NL4-3 recombinant viruses encoding wild-type subtype C Gag derived from patient SK-254 and SK-254 Gag with introduced subtype B-specific Gag residues 67S, 473P, and 483L/484Y. (C) Replication capacities of MJ4 recombinant viruses encoding wild-type subtype C Gag derived from patient SK-254 and SK-254 Gag with introduced subtype B-specific Gag residues 483L/484Y. Replication capacities were assayed in GXR cells using flow cytometry and are expressed relative to the capacity of the respective wild-type virus, which represents 100% replication. Bars represent the means from at least 3 independent experiments, and error bars represent standard deviations from the means.

FIG 6

Gag-protease subtype composition of the Kenyan Majengo cohort. (A) Maximum likelihood phylogenetic tree shows clustering of patient-derived Gag-protease gene sequences into distinct subtypes. Subtype reference A (A1, n = 37; A2, n = 20), C (n = 16), and D (n = 13) and intersubtype recombinant (n = 17) sequences are represented by black, red, purple, blue, and green, respectively. The scale bar indicates 2% nucleotide sequence divergence. (B) Illustration of Gag-protease intersubtype recombinants. The subtypes A, C, and D are represented by red, purple, and blue, respectively. Numbering is according to the HXB2 reference strain. Residues (150, 410, and 435) at which recombination breakpoints were common (observed 5 or more times) are highlighted in bold.

FIG 7

Intersubtype comparison of Gag-protease-mediated replication capacity in East African cohorts. (A) Significant differences in Gag-protease-mediated replication capacities of NL4-3 recombinant viruses encoding patient-derived Gag-proteases of subtypes A, C, and D and intersubtype recombinants from a Kenyan cohort. (B) Significant differences in replication capacities between subtypes are reproducible in a Ugandan cohort. Replication capacity was assayed in GXR cells using flow cytometry and normalized to the growth of the wild-type NL4-3 virus (replication capacity of 100%). Subtypes A, C, and D and intersubtype recombinants are represented in red, purple, blue, and green, respectively. The bars and whiskers represent the means and interquartile ranges, respectively. ANOVA (P value shown) with Tukey post hoc tests was used to test for significant differences between subtypes. The number of asterisks denotes the level of significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 8

Intersubtype comparison of Gag-driven replication capacity in PBMCs using the subtype C pZM246-F10 backbone. Comparison of the replication kinetics of pZM246-F10 (subtype C) viral constructs carrying patient-derived gag genes of different subtypes as well as NL4-3 gag. The patients for whom the derived Gag-protease NL4-3 recombinant viruses represented the average replication capacity for each subtype were selected for generation of patient-derived Gag pZM246-F10 recombinant viruses. Subtypes B, C, and D and intersubtype recombinants are represented in gray, purple, blue, and green, respectively. The replication assay was carried out in activated pooled PBMCs from two donors and monitored using p24 ELISA.