Cilloniz Cristian, Jackson Wallen, Grose Charles, Czechowski Donna, Hay John, Ruyechan William T
Department of Microbiology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, SUNY, Buffalo, NY 14214, USA.
J Virol. 2007 Jan;81(2):761-74. doi: 10.1128/JVI.01274-06. Epub 2006 Nov 1.
The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.
水痘带状疱疹病毒（VZV）的ORF9蛋白是疱疹病毒UL49基因家族的成员，但与UL49原型单纯疱疹病毒1型VP22的同源性和相似性有限。ORF9 mRNA是VZV感染期间表达量最高的信息；然而，关于ORF9蛋白的功能却知之甚少。我们发现VZV主要反式激活因子IE62和ORF9蛋白可从感染细胞中共沉淀出来。酵母双杂交分析将ORF9蛋白与IE62相互作用所需的区域定位在该蛋白中部的三分之一处，即包含氨基酸117至186的区域。用含有IE62 N端序列的GST-IE62融合蛋白进行的蛋白下拉试验表明，IE62酸性转录激活域的氨基酸1至43可与重组ORF9蛋白结合。对瞬时转染细胞的共聚焦显微镜观察表明，在没有其他病毒蛋白的情况下，ORF9蛋白定位于细胞质中，而IE62定位于细胞核中。在VZV感染的细胞中，ORF9蛋白定位于细胞质，而IE62则同时定位于细胞核和细胞质。共转染表达ORF9、IE62和病毒ORF66激酶的质粒导致ORF9和IE62在细胞质中显著共定位。用抗微管蛋白抗体进行的免疫共沉淀实验表明，感染细胞中存在ORF9-IE62-微管蛋白复合物。通过共聚焦显微镜观察到转染细胞中ORF9和微管蛋白的共定位。这些数据提示了一个ORF9蛋白功能模型，即在感染后期，ORF9蛋白在细胞质中与IE62以及可能的其他被膜蛋白形成复合物发挥作用。
