In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach

Abstract

The HIV-1 accessory protein Vpu exhibits high inter- and intra- subtype genetic diversity that may influence Vpu function and possibly contribute to HIV-1 pathogenesis. However, scalable methods to evaluate genotype/phenotype relationships in natural Vpu sequences are limited, particularly those expressing the protein in CD4+ T-cells, the natural target of HIV-1 infection. A major impediment to assay scalability is the extensive genetic diversity within, and immediately upstream of, Vpu's initial 5' coding region, which has necessitated the design of oligonucleotide primers specific for each individual HIV-1 isolate (or subtype). To address this, we developed two universal forward primers, located in relatively conserved regions 38 and 90 bases upstream of Vpu, and a single universal reverse primer downstream of Vpu, which are predicted to cover the vast majority of global HIV-1 group M sequence diversity. We show that inclusion of up to 90 upstream bases of HIV-1 genomic sequence does not significantly influence in vitro Vpu expression or function when a Rev/Rev Response Element (RRE)-dependent expression system is used. We further assess the function of four diverse HIV-1 Vpu sequences, revealing reproducible and significant differences between them. Our approach represents a scalable option to measure the in vitro function of genetically diverse natural Vpu isolates in a CD4+ T-cell line.

Patterns of genomic site inheritance in HIV-1M inter-subtype recombinants delineate the most likely genomic sites of subtype-specific adaptation

“… vpu Downregulation of CD4 and tetherin expression: this increases the pathogenesis of HIV-1M strains. Subtype C had the highest activity compared with Subtypes B and C ( Rahimi et al. 2017 ).…”

HLA-C downregulation by HIV-1 adapts to host HLA genotype

“…30% of the UARTO cohort samples included in this study were male and this cohort comprises predominantly heterosexual individuals. To characterize HLA-C downregulation a single plasma-derived Vpu sequence for 195 of these individuals, was cloned into a bicistronic pSel RRE –Vpu vector that expresses Vpu and GFP [ 70 ]. Of these 195 clones, Vpu sequences were of subtypes A (n = 45), B (n = 75), C (n = 19) or D (n = 56).…”

“…The 195 Vpu clones derived in pSel RRE –Vpu vectors, from chronically infected UARTO and BC HOMER cohort individuals, were assessed for ability to downregulate HLA-C in Molt-4 cells. Lipofectamine (ThermoFisher) was used according to manufacturer instructions to co-transfect ~200,000 cells with the pSel RRE –Vpu plasmid containing GFP and primary Vpu genes, and with the Rev expression plasmid pSel-Rev-ΔGFP previously described [ 70 ]. After 24hrs cells were stained with DT9 and an APC-conjugated secondary as above, and staining results were acquired using a SP6800 Spectral Cell Analyzer (Sony Biotechnology).…”

Unravelling the complicated evolutionary and dissemination history of HIV-1M subtype A lineages

“…For example, whereas the nef genes of subtype B isolates seem to downregulate CD4 and class I HLA allele expression more effectively than do the nef genes from subtype A, C, and D isolates ( Mann et al. 2013 ), the vpu genes from subtype C isolates tend to downregulate CD4 and tetherin more effectively than do the vpu genes of subtypes A, B, and A/D recombinant isolates ( Rahimi et al. 2017 ).…”