Mutagenesis and functional studies of the HIV-1 vpr gene and Vpr protein obtained from South African virus strains

[摘要] ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) isan accessory protein that interacts with a number of host cellular and other viralproteins. Vpr exerts several functions such as induction of apoptosis, induction of cellcycle G2 arrest, modulation of gene expression, and suppression of immuneactivation. The functionality of subtype C Vpr, especially South African strains, hasnot been studied. The aim of this study was to describe the diversity of South AfricanHIV-1 subtype C vpr genes and to investigate selected functions of these Vprproteins.Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, andsubtyped using phylogenetic analysis. Fragments containing natural mutations werecloned in mammalian expression vectors. A consensus subtype C vpr gene wasconstructed and site-directed mutagenesis was used to induce mutations in postions inwhich no natural mutations have been described. The functionality of all constructswas compared with the wild-type subtype B Vpr, by transfecting human 293T cellline to investigate subcellular localization, induction of apoptosis and cell cycle G2arrest. The modulation of genes expressed in the induction of apoptosis using TaqManLow density arrays (TLDA) was also investigated.Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conservedincluding motifs FPRPWL and TYGDTW, but the C-terminal was more variable. Thefollowing mutations were constructed using site-directed mutagenesis: P14I, W18C,Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vprlocalized to the nucleus but the W18C mutation disturbed the nuclear localization ofVpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, waslower than that of subtype B Vpr. All the natural mutants of subtype C Vpr inducedcell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycleG2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteinsinduced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells.However, an artificially designed Vpr protein containing the consensus sequences ofsubtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-CVpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants ofVpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associatedgene expression was similar for all constructs, indicated that apoptosis wasefficiently induced through the intrinsic pathway by the mutants.Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display asimilar ability for nuclear localization and apoptosis induction. The induction of cellcycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype CVpr proteins. The natural mutations studied in the isolates did not disturb thefunctions of subtype C Vpr and in some cases even potentiated the protein to induceapoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded asdefective, since enhanced functionality would be more indicative of an adaptive role.The increased potency of the mutated Vpr proteins suggests that Vpr may increase thepathogenicity of HIV-1 by adapting apoptotic enhancing mutations.

[发布机构] Stellenbosch University