一个新的膜转运介体PACS蛋白在疱疹病毒发生中的作用

Herpes simplex virus(HSV), the etiological agent of many human diseases, belongs to αsubfamily of Herpesviridae. During its replication, the nucleocapsids of herpes simplex virus are assembled in the nucleus of infected cells. How the nucleocapsids get teguments and envelope is still poorly understood and remains a contentious issue. One view is that the nucleocapsids get tegument in the nucleus, and by budding into perinuclear space, they obtain their envelope from the inner lamella of the nuclear membrane. Then the enveloped virions move through the endoplasmic reticulum and Golgi complex by vesicular transport, and finally are released out of the cell. The other view is that the nucleocapsids get transient envelopes from the inner lamella of the nuclear membrane, which will fuse with the membrane of the endoplasmic reticulum. This will release naked nucleocapsids into the cytosol. By budding into cytosolic membraneous compartments, most probably trans-Golgi network, the nucleocapsids are reenveloped. Several recent reports support the latter view. An implication of the latter view is that some envelope glycoproteins and tegument proteins must be localized on the membrane of the membraneous compartment so that the nucleocapsids can obtain these proteins whlie being enveloped in this compartment. It is well known that glycoprotein E of many α herpes viruses are localized to TGN, probably through the binding of their cytosolic acidic clusters to phosphofurin acidic cluster sorting protein 1(PACS-1). The aim of this study is to find if there are other proteins of herpes simplex virus localizing to TGN, clarify the mechanism of this localization, and establish the role of PACS-1 in the TGN localization of these proteins and in the biogenesis of HSV.First, we identified 33 candidate PACS-1 binding proteins in the proteome of HSV-1 which included some envelope glycoproteins and tegument proteins. Then we chose glycoprotein B to study its subcellular localization and its interaction with PACS-1. We first cloned the cytosolic domain of glycoprotein B of HSV-1 strain SM44(HSVgBcd) and functional domain of PACS-1(PACS-1fd) and expressed them in E.coli. Then we prepared antiserum against them. By double labeling immunofluorescence, HSV-1gB and PACS-1 were colocalized to TGN in BHK-21 cells infected by HSV-1 SM44. Rabbit antiserum against PACS-1fd could coimmunoprecipitate PACS-1 and HSV-1gB from the lysate of infected cells. GST pull-down assay showed that HSV-1gB and PACS-1fd could bind in vitro, and this binding was dependent on the phosphorylation of CKII phosphorylation site in the acidic cluster of HSV-1gBcd. Altogether the above results indicate that HSV-1gB is localized to TGN in infected cells and this localization is mediated by the interaction between the acidic cluster in its cytosolic domain and PACS-1. This suggests during the biogenesis of HSV, the nucleocasids in the cytosol obtain their envelopes by budding into TGN. It also suggest, by localizing some envelope glycoproteins(and possibly some tegument proteins) to TGN, PACS-1 plays an important role in the biogenesis of HSV.

本研究涉及体外测定疱疹病毒被膜糖蛋白与最新发现的膜蛋白转运介体PACS蛋白的结合反应，病毒被膜糖蛋白在培养细胞中的定位，以及观察PACS蛋白表达抑制对被膜糖蛋白细胞内分布的影响。进而评估这一膜蛋白转运介体在病毒发生的分子机制，并产生潜在的针对性靶蛋白以治疗由这类病毒引起的人类疾病。