Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 vif and inhibitors of Vif-APOBEC3G binding
Identification of an APOBEC3G binding site in human immunodeficiency virus type 1 vif and inhibitors of Vif-APOBEC3G binding
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DOI：
10.1128/jvi.00204-07
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发表时间：
2007-12-01
影响因子：
5.4
通讯作者：
Gabuzda, Dana
中科院分区：
文献类型：
--
作者：
Mehle, Andrew;Wilson, Heather;Gabuzda, Dana
APOBEC 3胞苷脱氨酶是限制人类免疫缺陷病毒1型（HIV-1）复制的有效抗病毒因子。HIV-1 Vif结合APOBEC 3G和APOBEC 3F，并通过与cullin 5和延伸蛋白B和C形成E3泛素连接酶靶向这些蛋白进行泛素化。Vif的N-末端区域是APOBEC 3G结合所必需的，但结合位点未知。为了鉴定Vif中的APOBEC 3G结合位点，我们以与高通量筛选的开发兼容的形式建立了可扩展的结合测定。使用重组蛋白的体外结合试验鉴定了抑制Vif-APOBEC 3G结合的Vif肽和单克隆抗体，并表明Vif残基33至83参与APOBEC 3G结合。基于细胞的结合测定证实了这些结果，并证明Vif的N末端的残基40至71含有APOBEC 3G的非线性结合位点。高度保守残基His 42/43的突变而非该区域中的其他带电残基的突变抑制Vif-APOBEC 3G结合、Vif介导的APOBEC 3G降解和病毒感染性。相比之下，这些残基的突变对Vif结合和APOBEC 3F的降解没有显著影响，表明Vif与APOBEC 3G和APOBEC 3F结合时对His 42/43的需求不同。这些结果鉴定了Vif的N末端中的非线性APOBEC 3结合位点，并证明针对该区域的肽或抗体可以抑制Vif-APOBEC 3G结合，从而验证Vif-APOREC 3界面作为潜在的药物靶标。
The APOBEC3 cytidine deaminases are potent antiviral factors that restrict replication of human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif binds APOBEC3G and APOBEC3F and targets these proteins for ubiquitination by forming an E3 ubiquitin ligase with cullin 5 and elongins B and C. The N-terminal region of Vif is required for APOBEC3G binding, but the binding site(s) is unknown. To identify the APOBEC3G binding site in Vif, we established a scalable binding assay in a format compatible with development of high-throughput screens. In vitro binding assays using recombinant proteins identified Vif peptides and monoclonal antibodies that inhibit Vif-APOBEC3G binding and suggested involvement of Vif residues 33 to 83 in APOBEC3G binding. Cell-based binding assays confirmed these results and demonstrated that residues 40 to 71 in the N terminus of Vif contain a nonlinear binding site for APOBEC3G. Mutation of the highly conserved residues His42/43 but not other charged residues in this region inhibited Vif-APOBEC3G binding, Vif-mediated degradation of APOBEC3G, and viral infectivity. In contrast, mutation of these residues had no significant effect on Vif binding and degradation of APOBEC3F, suggesting a differential requirement for His42/43 in Vif binding to APOBEC3G and APOBEC3F. These results identify a nonlinear APOBEC3 binding site in the N terminus of Vif and demonstrate that peptides or antibodies directed against this region can inhibit Vif-APOBEC3G binding, validating the Vif-APOREC3 interface as a potential drug target.