Architecture et Réactivité de l'ARN, Université de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084, Strasbourg cedex, France.
Nucleic Acids Res. 2010 Jan;38(2):633-46. doi: 10.1093/nar/gkp1009. Epub 2009 Nov 12.
The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.
HIV-1 病毒感染因子（Vif）通过拮抗细胞胞嘧啶脱氨酶 APOBEC-3G（hA3G）和 hA3F，允许非允许细胞（包括大多数天然 HIV-1 靶细胞）进行有效的感染。Vif 诱导这些限制因子通过蛋白酶体降解的机制已经得到了广泛研究，但对于 Vif 对 hA3G 和 hA3F 的翻译抑制作用知之甚少，该作用也被认为参与了 Vif 的功能。在这里，我们研究了 Vif 与 hA3G mRNA 的结合及其在翻译抑制中的作用。过滤结合实验和荧光滴定曲线显示，Vif 与 hA3G mRNA 紧密结合。Vif 对 3'UTR 的整体结合亲和力高于 5'UTR，尽管该区域至少包含一个高亲和力的 Vif 结合位点（表观 K(d) = 27 +/- 6 nM）。使用 RNase 足迹法在 5' 和 3'UTR 中鉴定了几个 Vif 结合位点。体外翻译实验表明，Vif 通过两种机制抑制 hA3G 的翻译：一种主要的非时间依赖过程需要 5'UTR，另一种额外的时间依赖、UTR 非依赖过程。使用在多聚化结构域中突变的 Vif 蛋白进行的结果表明，翻译控制的分子机制比简单的核糖体扫描物理阻断更为复杂。
