Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

Abstract

HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1 NL4.3 , and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag 147 , was identified in p24Gag (GQMVHQAI Gag140 -147 ). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-Nef E24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1 NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCEProtective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals

“…For each participant, three nef sequences were isolated from baseline plasma HIV RNA, cloned into a green fluorescent protein (GFP) reporter plasmid using previously described methods (36,38,45), and confirmed to cluster with their respective bulk plasma HIV RNA nef sequence (Fig. 1).…”

Nef-mediated inhibition of NFAT following TCR stimulation differs between HIV-1 subtypes

“…While Nef is a critical virulence factor in HIV infection (Kestler et al, 1991) and highly immunogenic (Radebe et al, 2011), it is also highly variable and largely excluded from the leading conserved T-cell based vaccine immunogens in testing (Borthwick et al, 2014;Mothe et al, 2015). Nevertheless, growing evidence suggests that a limited number of mutations in Nef, particularly specific mutation combinations, can impair the virus Shahid et al, 2015;Ueno et al, 2008), and that CTL targeting of selected Nef epitopes could lead to better clinical outcome (Adland et al, 2013). In the present analysis, two HLA-associated polymorphisms, 71K and 108D, were associated with decreased ability of Nef to inhibit TCR-mediated NFAT signalling.…”

Human leucocyte antigen but not KIR alleles and haplotypes associated with chronic HCV infection in a Chinese Han population

“…Shahid et al . demonstrated that HLA‐B*13 has a protective role for HIV infection by escape mutations on HIV‐1 replication and Nef function in a Caucasian population (Shahid et al, ). Zhang.…”

“…has not been reported as one of the genetic factors for HCV infection in previous studies, it has been reported to be associated with HIV infection. Shahid et al demonstrated that HLA-B*13 has a protective role for HIV infection by escape mutations on HIV-1 replication and Nef function in a Caucasian population(Shahid et al, 2015).Zhang. et al showed that HLA-A * 30-B * 13-C * 06 contributes to the slow progression of HIV infection in the northern Chinese.…”