The Research On Anti-HIV-1Activity Of APOBEC3G Mutant

AIDS is still a major threat to human health. Highly active anti-retroviral therapycan effectively control the replication of HIV-1. However, the high cost of treatment,viral resistance and drug toxicity hinder the treatment of HIV-1. There is an urgentneed to develop new anti-HIV-1drugs and treatment with novel targets.Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like3G(APOBEC3G, referred to as A3G) is an innate antiviral factor. HIV-1viral infectivityfactor (Vif) induced the degradation of A3G through ubiquitin-proteasome pathway toantagonize its anti-HIV-1activity. However, the interaction between A3G and Vif isspecies-specific, which is related to the residue at A3G128.Overlap PCR was adopted to construct D128K and D128R A3G mutants. In thisstudy, pEGFP-A3G D128K and pEGFP-A3G D128R eukaryotic plasmids expressinggreen fluorescent fusion protein were constructed, and then transfected into239FTand HeLa cells. Wild-type and mutated GFP-A3G fusion proteins were shown to belocalized in the cytoplasm using confocal laser scanning microscopy. Western-blotshowed that94.85%GFP-D128K and97.09%GFP-D128R protein was protectedfrom degradation by Vif, while real-time PCR demonstrated that Vif had no effects onA3G mRNA. Single round infection assay showed that the inhibitive rates ofwild-type and mutant of A3G were both above90%when higher dose was used.When the dose was reduced, the inhibitive rates of wild-type A3G were dropped to25%~38%while the inhibitive rates of A3G mutants were still80%~90%. When0.001μg or less A3G was used, both wild-type and mutants lost the ability to inhibitHIV-1infection. This study showed that the A3G mutants can resist the degradation byVif, thus inhibiting HIV-1infection.