Pol-Driven Replicative Capacity Impacts Disease Progression in HIV-1 Subtype C Infection

Abstract

CD8⁺ T cell-mediated escape mutations in Gag can reduce HIV-1 replication capacity (RC) and alter disease progression, but less is known about immune-mediated attenuation in other HIV-1 proteins. We generated 487 recombinant viruses encoding RT-integrase from individuals with chronic (n = 406) and recent (n = 81) HIV-1 subtype C infection and measured their in vitro RC using a green fluorescent protein (GFP) reporter T cell assay. In recently infected individuals, reverse transcriptase (RT)-integrase-driven RC correlated significantly with viral load set point (r = 0.25; P = 0.03) and CD4⁺ T cell decline (P = 0.013). Moreover, significant associations between RT integrase-driven RC and viral load (r = 0.28; P < 0.0001) and CD4⁺ T cell count (r = -0.29; P < 0.0001) remained in chronic infection. In early HIV infection, host expression of the protective HLA-B*81 allele was associated with lower RC (P = 0.05), as was expression of HLA-B*07 (P = 0.02), suggesting early immune-driven attenuation of RT-integrase by these alleles. In chronic infection, HLA-A*30:09 (in linkage disequilibrium with HLA-B*81) was significantly associated with lower RC (P = 0.05), and all 6 HLA-B alleles with the lowest RC measurements represented protective alleles, consistent with long-term effects of host immune pressures on lowering RT-integrase RC. The polymorphisms V241I, I257V, P272K, and E297K in reverse transcriptase and I201V in integrase, all relatively uncommon polymorphisms occurring in or adjacent to optimally described HLA-restricted cytotoxic T-lymphocyte epitopes, were associated with reduced RC. Together, our data suggest that RT-integrase-driven RC is clinically relevant and provide evidence that immune-driven selection of mutations in RT-integrase can compromise RC.IMPORTANCE Identification of viral mutations that compromise HIV's ability to replicate may aid rational vaccine design. However, while certain escape mutations in Gag have been shown to reduce HIV replication and influence clinical progression, less is known about the consequences of mutations that naturally arise in other HIV proteins. Pol is a highly conserved protein, but the impact of Pol function on HIV disease progression is not well defined. Here, we generated recombinant viruses using the RT-integrase region of Pol derived from HIV-1C-infected individuals with recent and chronic infection and measured their ability to replicate in vitro We demonstrate that RT-integrase-driven replication ability significantly impacts HIV disease progression. We further show evidence of immune-mediated attenuation in RT-integrase and identify specific polymorphisms in RT-integrase that significantly decrease HIV-1 replication ability, suggesting which Pol epitopes could be explored in vaccine development.

Keywords: HIV-1 subtype C; HLA polymorphisms; replication capacity.

FIG 1

Phylogenetic relatedness and diversity of the RT-integrase sequences from bulk plasma and matched recombinant viruses. (A) A maximum likelihood tree showing the phylogenetic relatedness of HIV-1 subtype C RT-integrase sequences from the plasma and respective recombinant viruses at a scale of 0.02. The prefix RI indicates sequences derived from recently infected individuals, while the rest of the sequences were derived from chronically infected individuals. Blue indicates the bulk plasma sequences, while red indicates the recombinant virus sequences. (B) The mean number of nucleotide mixtures in plasma sequences derived from chronic infection (mean, 17; standard deviation, 13) compared to their respective recombinant virus sequences (mean, 6; standard deviation, 6) is shown. This indicates reduced diversity in the recombinant virus sequences (paired t test). For example, for participant SK313, there were 6 full nucleotide differences between the plasma and virus sequences and the number of mixtures in the plasma and virus sequences was 17 and 6, respectively.

FIG 2

Relationship between Gag-protease- and RT-integrase-mediated RC. (A) The distributions of RT-integrase-driven RC for HIV-1C recent and chronic infection are shown. (B) The RC of NL4-3 recombinant viruses encoding participant-derived RT-integrase and those encoding Gag-protease from the same individuals were significantly correlated (P = 0.0002 by Pearson's correlation).

FIG 3

Length of RT-integrase sequence impacts RC. NL4-3 recombinant viruses encoding participant-derived RT-integrase sequences of 866 amino acids (AA) had a lower RC than those encoding participant-derived RT-integrase sequences of the same length as HXB2 (849 AA) (P = 0.0047 by Student's t test).

FIG 4

Relationship between RT-integrase-mediated RC in early infection and markers of disease progression. (A) The RC of the recombinant viruses encoding RT-integrase derived from individuals with early HIV-1 C infection correlated negatively with subsequent viral load set point (P = 0.03 by Spearman's correlation). (B) Higher RC corresponds to a higher rate of CD4 decline. The regression lines show prediction of CD4 decline when RT-integrase RC is stratified by the median as RC of >0.92 (red) and RC of <0.92 (blue) (P < 0.001 by GEE model). The bars represent 95% confidence intervals. Sqrt, square root.

FIG 5

Relationship between RT-integrase-mediated RC in chronic infection and markers of disease progression. The RC of NL4-3 recombinant viruses encoding RT-integrase derived from chronically HIV-1C-infected individuals correlated positively with the plasma log viral load (A) and negatively with the CD4 count (B). Pearson's (P < 0.0001) and Spearman's (P < 0.0001) correlations were used, respectively.

FIG 6

Associations between HLA class I allele expression and RT-integrase RC. The RC of recent (A to C) and chronic (D to F) viruses were stratified by expression of the host HLA-A (A and D), HLA-B (B and E), and HLA-C (C and F) alleles. The box plots display RC results arranged by lowest mean RC at the bottom and the highest mean RC at the top. Dots indicate the means, and vertical lines indicate the medians. Boundaries of the boxes indicate the interquartile ranges, while the whiskers display the maximum and minimum viral RC values. The continuous vertical line on each graph indicates the mean RC for each cohort. HLA-I alleles with a minimum of n = 5 are shown. Asterisks indicate HLA alleles that are significantly associated with either higher or lower RC (Student's t test).

FIG 7

Relationships between HLA-associated polymorphisms in RT or integrase and RT-integrase-mediated RC in early infection. The number of RT HLA-associated polymorphisms correlated positively with RC (P = 0.05 by Spearman's correlation) (A), and the association was stronger when considering only those polymorphisms present in or adjacent to A list epitopes (P = 0.02 by Student's t test) (B). The number of integrase polymorphisms correlated negatively with RC (P = 0.024 by Spearman's correlation) (C), and the association was strengthened when limited to those polymorphisms present in or adjacent to A list epitopes (P < 0.0001 by ANOVA) (D). Counts were performed irrespective of the host HLA alleles.