Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes

Abstract

BackgroundThe highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef’s most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D.ResultsSingle HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function.ConclusionsResults support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

Impaired Nef Function Is Associated with Early Control of HIV-1 Viremia

“…As expected for samples obtained during early infection following peak viremia, pVL correlated inversely with the estimated number of days postinfection in both AC (Spearman R ϭ Ϫ0.77, P ϭ 0.01) and AP (R ϭ Ϫ0.38, P ϭ 0.007). Consistent with previous observations in other cohorts (21,56,57), Nef CD4 and HLA class I downregulation functions correlated positively in both AC (R ϭ 0.74, P ϭ 0.02) and AP (R ϭ 0.56, P Ͻ 0.001). Given that these activities are largely genetically separable in mutational studies of HIV-1 reference strains (58,59), this result supports the idea of the existence of secondary genetic determinants lying outside critical motifs in patient-derived Nef isolates that may modulate function and/or protein stability.…”

“…Similarly, it has been reported that the effects of Nef on host HLA expression may differ between HLA alleles (82). While we assessed only downregulation of HLA-A*02 here, a previous study by our group observed a strong correlation between the abilities of patient-derived Nef clones to downregulate A*02 and B*07 in this same assay system (56). Changes in Nef function between baseline and follow-up clones sampled were observed up to 516 days later.…”

“…For each AC, the function of 2 to 5 unique clones was assessed. Results were normalized to those of a positive control (SF2 Nef ) that displays strong CD4 and HLA class I downregulation activities (21,56) such that values less than 1.0 indicate lower Nef function and values greater than 1.0 indicate higher Nef function relative to SF2 Nef . A negative-control plasmid lacking nef did not downregulate either surface protein (Fig.…”

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

“…The importance of HIV-mediated CD4 down-modulation for the viral replication and the disease progression [2][3][4]20] renders research on this topic of vital importance to better understand the virus and to identify host factors involved in the process. We were especially interested in exploring the cellular pathways that are exploited by the virus to achieve CD4 down-modulation.…”

The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation

“…Human immunodeficiency virus type 1 (HIV-1)-induced CD4 downregulation is an important determinant of viral replication in vivo (Ali et al, 2009;Argañaraz et al, 2003;Carl et al, 2001;Casartelli et al, 2003;Mann et al, 2013Mann et al, , 2014Watkins et al, 2013) and for years it has been proposed as an interesting target for the research of new antiviral therapeutic strategies. Cellular co-factors of HIV could have great potential in the future of anti-retroviral therapy (Arhel & Kirchhoff, 2010;Tintori et al, 2014); therefore it is important to completely elucidate the CD4 downregulation pathway in order to find possible co-factors involved in the process.…”