Wichroski Michael J, Ichiyama Kozi, Rana Tariq M
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
J Biol Chem. 2005 Mar 4;280(9):8387-96. doi: 10.1074/jbc.M408048200. Epub 2004 Nov 10.
To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When co-expressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif(C114S), which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif(C114S) from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile(9) disrupted the ability of Vif or Vif(C114S) to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo.
为研究HIV-1病毒感染性因子（Vif）如何介导蛋白酶体依赖性宿主因子载脂蛋白B mRNA编辑酶催化多肽样3G（APOBEC3G）的消耗，将功能性和非功能性Vif-APOBEC3G相互作用与亚细胞定位相关联。APOBEC3G定位于整个细胞质，并与γ-微管蛋白、20S蛋白酶体亚基和泛素在点状细胞质小体中共定位，这些小体可用于监测细胞中Vif-APOBEC3G的相互作用。通过免疫染色和实时成像，我们发现相当一部分Vif定位于细胞核，并且这种定位因氨基酸12-23的缺失而受损。当共表达时，Vif在细胞质中的定位更为明显，并降低了细胞内APOBEC3G的总水平，但很少与APOBEC3G在细胞质小体中共定位。相反，无活性但继续与APOBEC3G相互作用的Vif（C114S）在细胞质中与APOBEC3G稳定结合，导致在细胞质小体中完全共定位，并使Vif（C114S）从细胞核中呈剂量依赖性排除。蛋白酶体抑制后，细胞质中APOBEC3G水平升高，两种蛋白非特异性地共同积聚到波形蛋白包裹的聚集体中。此外，无论有无APOBEC3G，蛋白酶体抑制均显著改变Vif的定位，表明异常定位也可能导致Vif功能丧失。最后，Vif异亮氨酸（9）处的突变破坏了Vif或Vif（C114S）与APOBEC3G共免疫沉淀和共定位的能力，表明Vif的N末端介导与APOBEC3G的相互作用。综上所述，这些结果表明细胞质中Vif-APOBEC3G相互作用是Vif调节APOBEC3G所必需的，但并不充分，并且可以通过体内共定位来监测。
