ROLE OF GM IN KSHV ENTRY, EGRESS AND VIRUS-INDUCED CELL FUSION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。卡波西肉瘤相关疱疹病毒(KSHV)，又称人类疱疹病毒8型，属于伽马疱疹病毒亚科，被认为是卡波西肉瘤、原发性渗出性淋巴瘤和多中心Castleman病的一个亚型的病原体。KSHV类似于疱疹病毒家族中的其他病毒，指定在病毒生命周期中表达的多种糖蛋白。这些KSHV糖蛋白中的许多已被证明在病毒进出和病毒诱导的细胞融合中起重要作用。糖蛋白M(GM)和gn在疱疹病毒的多个步骤中起重要作用，但它们在KSHV感染中的潜在作用尚不清楚。这个项目的中心假设是KSHV GM和GN在病毒生命周期的两个关键方面发挥重要作用：a)细胞质病毒粒子的形成和出口；b)病毒诱导的细胞融合。研究的总体实验方法是利用克隆到细菌人工染色体(BAC)中的KSHV基因组来产生缺乏糖蛋白M(GM)或糖蛋白N(Gn)基因的KSHV重组病毒。这些病毒将被用来研究这两种糖蛋白在病毒粒子形态发生和出口中的功能，以及由在GB中具有合胞突变的KSHV突变病毒引起的病毒诱导的细胞融合。特异性目的一：利用DH10B大肠杆菌中pGET-Rec重组系统构建KSHV-BAC36 Delta Gm(Gm-Null)和BAC36 Delta gn(Gn-Null)突变体，并对其进行遗传学鉴定。特异性目标II：评估KSHV Gm和Gn在细胞质病毒粒子形态发生和出口以及病毒诱导细胞融合中的作用

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, belongs in the gammaherpesvirinae subfamily and is believed to be the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman's disease. KSHV, similar to the other viruses in the herpesvirus family, specifies numerous glycoproteins which are expressed during the virus lifecycle. Many of these KSHV glycoproteins have been shown to be important in virus entry, egress and virus-induced cell fusion. Glycoprotein M (gM) and gN are known to play important roles in multiple steps of herpesviruses, while their potential roles in KSHV infections are unknown. The central hypothesis of this project is that the KSHV gM and gN serve essential roles in two critical facets of the viral lifecycle: a) cytoplasmic virion morphogenesis and egress; b) virus-induced cell fusion. The overall experimental approach of the proposed investigations is to generate KSHV recombinant viruses deficient in either glycoprotein M (gM) or glycoprotein N (gN) genes utilizing the KSHV genome cloned into a bacterial artificial chromosome (BAC). These viruses will be utilized to address the functions of these two glycoproteins in virion morphogenesis and egress, and virus-induced cell fusion caused by a KSHV mutant virus having syncytial mutations in gB. Specific Aim I: To construct and genetically characterize KSHV-BAC36 delta gM (gM-null) and BAC36 delta gN (gN-null) mutants utilizing the pGET-Rec recombination system in DH10B E. coli Specific Aim II: To assess the role of KSHV gM and gN in cytoplasmic virion morphogenesis and egress, and virus-induced cell fus