The Role Of PACS-1 In Trans-Golgi Network Localization Of Herpes Simplex Virus 1 Glycoprotein B

Herpes simplex virus( HSV), the etiological agent of many human diseases, belongs to a subfamily 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 rraw-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 a herpes viruses are localized to TON, probably through the binding of their cytosolic acidic clusters to phosphofurin acidic cluster sorting protein l(PACS-l). The aim of this study is to find if there are other proteins of herpes simplex virus localizing to TON, clarify the mechanism of this localization, and establish the role of PACS-l in the TGN localization of these proteins and in the biogenesis of HSV.First, we screened candidate PACS-l binding proteins in the proteome ofHSV-1 strain 17 by sequence analysis according to the characteristics of the reported PACS-l binding acidic clusters. The result showed that among all 71 HSV-1 strain 17 proteins in SWISS-PROT databank, 33 were candidate PACS-l binding proteins, which included some envelope glycoproteins and tegument proteins.In order to verity the proteins in these 33 candidates that can actually bind with PACS-l and localize to TGN, we chose glycoprotein B to study its subcellular localization and its interaction with PACS-I. By RT-PCR, we first cloned the cytosolic domain of glycoprotein B of HSV-1 strain SM44(HSVgBcd). Sequence analysis showed that the amino acids of the cytosolic domain of HSV-1 glycoprotein B were highly conserved between different strains. There are 1 nucleus localization signal, two tyrosine-based motif and one acidic cluster containing casein kinase II(CKII) phosphorylation site in the cytosolic domain. Then we cloned functional domain of PACS-l(PACS-lfd) from the total RNA of SD rat brain by RT-PCR. The sequencing results showed that the sequence of our PACS-lfd clone was 100% identical with the reported sequence. Afterwards, we expressed two fusion proteins,GST/HSVgBcd and GST/PACS-lfd, in E. coli BL21 by GST expression system. First we optimized the conditions of protein expression via orthogonal experiment design. Then we prepared GST/HSVgBcd and GST/PACS-lfd in large scale under optimized conditions. GST/HSVgBcd mainly presented as inclusion body. After solubilization and refolding of inclusion body, the protein concentration of the preparation was 0.165mg/ml and the purity of GST/HSVgBcd was 32.188%. After purification and thrombin cleavage, the prepared GST/PACS-lfd had a protein concentration of 0.2-0.37mg/ml and the purity of GST/PACS-lfd was 47.588%. Then we prepared murine antiserum against GST/HSVgBcd, the titer of which ^ 1 '. 400. We also prepared rabbit antiserum against PACS-lfd with a titer ^ 1 '. 3200. Immunofluorescence and Western blot indicated that the two antiserum were specific and could recognize their antigens in both natural and denatured conformation. By double labeling immunofluorescence, HSV-1 gB and PACS-l were colocalized to TGN in BHK-21 cells infec...