Sequence diversity of HIV-1 subtype C accessory genes VIF, VPR and VPU

Abstract

OBJECTIVES: To date there is no effective and safe vaccine to stop the spread of human immunodeficiency virus (HIV) and provide cross protection among different subtypes. HIV accessory genes were overlooked for many years and recently they are becoming candidates for development of new anti-HIV drugs and vaccines. This is supported by their ability to elicit cytotoxic T lymphocyte response. To date, there are limited studies on accessory genes (nef, vif, vpr and vpu) on South African HIV strains. This study sought to amplify and analyse the sequences of HIV-1 subtype C accessory genes (vif, vpr and vpu) to assess the genetic diversity as well as the motifs and residues associated with key biological functions of these genes. This study further sought to compare the degree of genetic diversity between the accessory and structural genes. METHODS: The study was an exploratory study using stored (-70ºC) HIV positive plasma samples. The study population comprised of 25 HIV positive plasma samples which were already sequenced in the gag and env genes in another study. The samples were drawn from the neighbouring townships of Pretoria: Ga-Rankuwa, Soshanguve, Mamelodi, Laudium, Kalafong, Jubilee and Mabopane. For the purpose of this study, the same samples were amplified, sequenced and characterised in the pol and accessory (vif, vpr and vpu) genes in order to obtain near full length sequences of the HIV isolates from Pretoria region. Six samples were cloned for accessory genes. Five clones from each sample were selected. Sequence analysis was performed for all the PCR amplicons and clones. Base calling for the sequences generated was performed on Chromas Pro program. Computing of phylogenetic tree was performed with MEGA 4 program. ClustalW software was used for sequence alignment and translation of nucleotides to amino acids was performed with BioEdit. The amino acid alignments were analysed on graphic view. RESULTS: All 25 samples were successfully amplified for accessory genes (vif, vpr and vpu) and pol gene. All the 25 pol PCR amplicons were successfully sequenced, while all but one accessory PCR amplicons were successfully sequenced. A number of conserved motifs and residues were observed in all the four genes (vif, vpr, vpu and pol). Vif and vpr showed to harbour most of these conserved motifs and residues; 144-SLQYLA-149 and H71 respectively. In addition, the R77Q mutation associated with long term non-progressors was observed in the vpr gene of 15 sequences. Drug resistant mutations were evaluated in both protease and RT regions. Nine samples had one or two drug resistant mutations i.e T74S, L10I, V179D, E138A/D, Y318F,Y181C and K108N. Phylogenetic analysis confirmed the 25 HIV positive samples to be HIV-1 subtype C in both structural and accessory genes. The genetic diversity of HIV-1 subtype C was compared between accessory (vif, vpr and vpu) and structural (pol, gag and env) genes. The gag and env sequences were available from a previous project (Musyoki, 2009). The gag and vif gene sequences were highly conserved (89% to 96% and 88% to 96%, respectively), as compared to vpr gene (84% to 94%), the pol gene (79% to 95%), the env gene (83% to 93%) and finally the vpu gene (73% to 92%). CONCLUSION: This study found that amplification of clones was more sensitive as compared to direct samples and analysis of clone sequences was more clear than analysis of direct PCR products. Functional motifs and residues observed in all accessory genes were highly conserved. Vif was more conserved, followed by vpr and vpu, respectively. Genetic analysis of pol gene revealed that there were drug resistant strains in circulation. This indicates that the patients were infected with drug resistant viruses; this cannot be verified from the study population. And that most of the strains in this study had mutations associated with long term non-progressors (LTNP’s). However, it is not known whether these patients were indeed LTNP’s. Comparison of genetic diversity between structural and accessory genes demonstrated that, gag, vif and vpr were more conserved than pol, env and vpu.