The Vaccinia Virus E8R Gene Product: a Viral Membrane Protein That Is Made Early in Infection and Packaged into the Virions' Core

Vaccinia virus (VV), a member of the poxvirus family, is unique among most other DNA viruses in that both transcription and DNA replication occur in the cytoplasm of the host cell. It was recently shown by electron microscopy (EM) that soon after viral DNA synthesis is initiated in HeLa cells, the replication sites become enwrapped by the membrane of the endoplasmic reticulum (ER). In the same study, a novel VV membrane protein, the E8R gene product, that may play a role in the ER wrapping process was identified (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001). In the present study, the gene product of E8R was characterized both biochemically and morphologically. We show that E8R is made predominantly early in infection but is packaged into the virion. On two-dimensional gel electrophoresis, the protein appeared as a single spot throughout the VV life cycle; however, in the assembled virion, the protein underwent several modifications which resulted in a change in its molecular weight and its isoelectric point. EM of labeled cryosections of infected HeLa cells showed that the protein localized to the ER and to membranes located on one side of the Golgi complex as early as 1 h postinfection. Late in infection, E8R was additionally associated with membranes of immature virions and with intracellular mature viruses. Although E8R is predominantly associated with membranes, we show that the protein is associated with viral cores; the protein is present in cores made with NP-40-dithiothreitol as well as in incoming cores, the result of the viral entry process, early in infection. Finally, we show that E8R can be phosphorylated in vitro by the viral kinase F10L. It is able to bind DNA in vitro, and this binding may be modulated by phosphorylation by F10L. A putative role of the E8R gene product throughout the VV life cycle is discussed.痘病毒科成员牛痘病毒（Vaccinia virus, VV）与绝大多数其他DNA病毒存在显著差异，其转录与DNA复制过程均发生于宿主细胞的细胞质内。近期借助电子显微镜（electron microscopy, EM）的研究表明，在海拉细胞（HeLa cells）中启动病毒DNA合成后不久，病毒复制位点便会被内质网（endoplasmic reticulum, ER）膜所包裹。在该项研究中，研究者还鉴定出一种新型VV膜蛋白——E8R基因编码产物，其可能参与内质网膜包裹过程（N. Tolonen、L. Doglio、S. Schleich及J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001）。本研究从生化与形态学两个维度对E8R基因编码产物进行了系统表征。研究证实，E8R蛋白主要在感染早期合成，并被包装进入成熟病毒粒子中。在二维凝胶电泳（two-dimensional gel electrophoresis）实验中，该蛋白在VV整个生命周期内均以单一斑点形式存在；但在组装完成的病毒粒子中，该蛋白会发生多种翻译后修饰，导致其分子量与等电点（isoelectric point）发生改变。对感染海拉细胞的标记冷冻切片进行电子显微镜观察后发现，早在感染后1小时，E8R蛋白便定位于内质网以及高尔基体单侧的膜结构中。感染后期，E8R还会与未成熟病毒粒子的膜结构以及细胞内成熟病毒粒子相结合。尽管E8R蛋白主要与膜结构结合，本研究同样证实该蛋白可与病毒核心相结合：无论是经NP-40-二硫苏糖醇（dithiothreitol, DTT）处理得到的病毒核心，还是感染早期病毒侵入宿主过程中产生的侵入性病毒核心，均含有该蛋白。最后，本研究证实E8R可在体外被病毒激酶F10L磷酸化。该蛋白在体外能够结合DNA，且这种DNA结合活性可被F10L介导的磷酸化所调控。文末还讨论了E8R基因编码产物在VV整个生命周期中潜在的生物学功能。