Study of anti-APOBEC3G activities of HIV-1 Vif proteins among different subtypes

抗逆转录病毒胞苷脱氨酶APOBEC3G在外周血淋巴细胞和巨噬细胞中大量表达，强烈保护这些细胞免受HIV-I感染。HIV-1的Vif蛋白通过诱导蛋白酶体介导的APOBEC3G降解来克服这种抗病毒作用，是维持病毒感染性的关键。VIF基因全长579个碱基，在HIV-I亚型中表现出高度的遗传多样性，因此，研究来自不同亚型的VIF蛋白对APOBEC3G的病毒防御活性是否存在差异，是一个有趣的问题。为了测试这一点，我们创建了编码来自A、B、C、CRF01_AE和CRF02_AG临床分离株的Vif蛋白的表达载体，并比较了它们的抗APOBEC3G活性。在不同亚型的APOBEC3G和Vif蛋白存在下从细胞中产生的病毒，表现出不同的病毒感染性，即C亚型Vif蛋白与任何亚型的Vif蛋白相比，都显示出高的抗APOBEC3G活性。其他亚型衍生的Vif蛋白，这被发现是由于Vif对蛋白酶体APOBEC3G的降解程度不同，取决于亚型。为了确定C亚型Vif蛋白的哪一部分将负责ANIT-APOBEC3G的强大活性，我们在B和C亚型之间创建了嵌合Vif结构，并通过产生Vif质粒的点突变来进一步寻找负责的氨基酸(S)。结果，我们在N-末端鉴定了两个氨基酸，它们与C-Vif亚型特异性抗APOBEC3G活性有关。有趣的是，这些氨基酸中的一个包含在保守序列中，被认为参与了与APOBEC3G的相互作用，这表明C-VIF亚型可能比其他亚型的Vif蛋白更有效地结合APOBEC3G。这些结果表明，不同亚型Vif蛋白的生物学差异可能对病毒的传播性产生影响。

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-I infection. The Vif protein of HIV-1 overcomes this antiviral effect by inducing proteasome-mediated degradation of APOBEC3G, and is a key for maintaining viral infectivity. The vif gene, which is 579-bp long, displays high genetic diversity among HIV-I subtypes and it is therefore intriguing to address whether Vif proteins derived from different subtypes might be differ in the viral defense activity against APOBEC3G. To test this, we created expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG clinical isolates and compared their anti-APOBEC3G activities. Viruses, produced from cells in the presence of APOBEC3G and Vif proteins of different subtypes, showed differential viral infectivities, that is, subtype C-derived Vif proteins showed exclusively high anti-APOBEC3G activities, compared with any. other subtype-derived Vif proteins, and this was found to be because of the different levels of proteasomal APOBEC3G degradation by Vif, depending on subtypes. To determine which portion of subtype C-Vif protein would be responsible for the robust anit-APOBEC3G activities, we created chimeric Vif constructs between subtypes B and C, and further pursued the responsible amino acid (s) by generating point mutants of Vif plasmids. As a result, we identified two amino acids at N-terminus responsible for subtype C-Vif-specific anti-APOBEC3G activity. Intriguingly, one of these amino acids is contained in the conserved sequence which is proposed to be involved in the interaction with APOBEC3G, suggesting that subtype C-vif might be able to bind APOBEC3G more efficiently than do Vif proteins of other subtypes. These results imply that biological differences of Vif proteins among subtypes might have an impact on viral transmissibility.