The role of Nef-mediated SERINC5 down-regulation on HIV-1 disease progression.

Abstract

HIV-1 Nef is a small accessory protein that plays a vital role in enhancing HIV-1 pathogenesis, evidenced by a strongly attenuated disease course following infection with a virus with gross Nef defects. Nef has multiple cellular effects, which enhance HIV-1 replication and immune evasion. Major activities of Nef include CD4 down-regulation, HLA-I down-regulation, and CD4-independent enhancement of virion infectivity. Recent studies have uncovered Nefmediated down-regulation of the host restriction factor SERINC5 as an important mechanism by which Nef enhances virion infectivity. However, there is a lack of studies defining the role of this function in HIV-1 pathogenesis. Previous studies indicated that Nef-mediated CD4 down-regulation and enhancement of infectivity are likely the major contributors to Nef’s effect of enhancing pathogenicity; the relative significance of each Nef function for HIV-1 disease progression remains incompletely understood. Given the key role of Nef-mediated SERINC5 down-regulation in enhancing virion infectivity, the primary aim of the present study was to determine if this Nef activity contributes significantly to disease progression in individuals infected with HIV-1 subtype C, which is the dominant HIV-1 subtype worldwide. To investigate this, SERINC5 down-regulation activity of 106 Nef clones derived from patients with early HIV-1 subtype C infection were evaluated in a CD4+ T cell line using a flow cytometry-based assay and subsequently related to viral load set point and to the rate of CD4+ T cell decline using linear regression analysis. The second aim of this study was to assess the overall contribution of SERINC5 down-regulation to Nef function, using linear regression analysis with E values as a proxy for overall Nef function in vivo.

The third aim of the study was to identify amino acid variants that significantly alter Nefmediated SERINC5 down-regulation using a codon-by-codon sequence-function analysis tool available online. No significant relationship was found between each Nef function and viral set point (SERINC5 down-regulation, p=0.28) or rate of CD4+ T cell decline (SERINC5 down-regulation, p=0.48). CD4 down-regulation (p=0.02) and SERINC5 down-regulation (p=0.003) were significant determinants of the E value in univariate analyses, and SERINC5 down-regulation remained significant in the multivariate analysis (p=0.003). We found several amino acids that were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. In conclusion, none of the Nef functions in our study, including SERINC5 down-regulation, were found to be significant individual contributors to disease progression. However, interestingly we found CD4 down-regulation and SERINC5 down-regulation to be the largest contributors, of the Nef functions considered here, to overall Nef function and that the contribution of SERINC5 down-regulation was the most significant. Taken together, this could be explained by multiple Nef functions acting together to facilitate the enhancement of viral spread and immune evasion in vivo that ultimately enhance disease progression. We found several amino acid variants that either increased or decreased Nef’s ability to down-regulate SERINC5; however, further studies in the form of site-directed mutagenesis are warranted to further understand their effect on SERINC5 down-regulation activity. In summary, the results suggest that SERINC5 down-regulation is a strong contributor to overall Nef function and identifies potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.