GENETICS AND FUNCTIONS OF KSHV GLYCOPROTEINS GM AND GN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 卡波西肉瘤相关疱疹病毒（KSHV）或人类疱疹病毒8（一种嗜淋巴细胞致癌病毒）与卡波西肉瘤、体腔B细胞淋巴瘤（BCBL）或原发性渗出性淋巴瘤以及某些形式的多中心Castleman病的发病机制有关。与其他疱疹病毒相似，KSHV编码许多糖蛋白，这些糖蛋白在病毒粒子的形态发生和感染性中起重要作用。本研究的总体目标是研究KSHV糖蛋白在病毒生命周期中的结构和功能，最初的重点是糖蛋白gM和gB。已经使用两种主要方法来阐明糖蛋白gM和gB的结构和功能：1）通过使用克隆到细菌人工染色体（bac）中的KSHV基因组构建KSHV突变体; 2）使用siRNA有条件地沉默病毒糖蛋白表达。通过在大肠杆菌中将gM基因插入KSHV-bac 36基因组中，构建了gM缺失的KSHV突变病毒。杆菌 将KSHV gM-无效病毒基因组转染到293细胞中产生病毒粒子，其能够从转染的细胞中流出并感染其他细胞，表明gM对于病毒粒子流出和感染性并不重要。已知KSHV gB在病毒体外出中是重要的。我们对gB的研究主要集中在gB胞质末端在病毒诱导的细胞融合和病毒体逃逸中的作用。突变KSHV病毒构建指定gB羧基末端缺失。25个氨基酸的gB缺失显著增加了病毒诱导的细胞融合。为了使用在诱导病毒裂解复制时产生大量病毒的BCBL-1细胞系描绘涉及病毒体排出的gB的功能结构域，使用了gB特异性siRNA。 siRNA转染入BCBL-1细胞显著减少了病毒体从BCBL-1细胞中的排出。 通过抗gB siRNA抑制BCBL-1细胞的转染，其中密码子优化的gB拯救了病毒粒子排出。类似地，用表达gB-截短的质粒转染挽救了病毒体排出，表明gB的胞质末端对于病毒体排出并不重要。

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), or human herpesvirus 8, a lymphotropic oncogenic virus, has been implicated in the pathogenesis of Kaposi's sarcoma; body cavity-based B-cell lymphoma (BCBL), or primary effusion lymphoma; and some forms of multicentric Castleman's disease. Similar to other herpesviruses, KSHV encodes many glycoproteins, which play important roles in virion morphogenesis and infectivity. The overall goal of this research is to investigate the structure and function of KSHV glycoproteins in the virus life-cycle with an initial focus on glycoproteins gM and gB. Two main approaches have been used to elucidate the structure and function of glycoproteins gM and gB: 1) Construction of KSHV mutants through the use of the KSHV genome cloned into an bacterial artificial chromosome (bac); 2) Conditional silencing of viral glycoprotein expression using siRNAs. gM-null KSHV mutant viruses were constructed by insertional inactivation of the gM gene into the KSHV-bac36 genome in E. coli. Transfection of the KSHV gM-null viral genome into 293 cells produced virions, whcih were able to egress out-of-the transfected cells and infect other cells indicating that gM was not important for virion egress and infectivity. KSHV gB is known to be important in virion egress. Our work on gB focused on the role of the gB cytoplasmic terminus in virus-induced cell fusion and virion egress. Mutant KSHV viruses were constructed that specified gB carboxyl terminal deletions. A gB deletion of 25 amino acids substantially increased virus-induced cell fusion. To delineate functional domains of gB involved in virion egress using the BCBL-1 cell line that produces large amounts of virus upon induction of the virus to lytic replication, siRNAs specific for gB were utilized. Transfection of siRNAs into BCBL-1 cells significantly reduced virion egress from BCBL-1 cells. Tranfection of BCBL-1 cells inhibited by anti-gB siRNAs with a codon optimized gB rescued virion egress. Similarly, transfections with plasmids expressing gB-truncations rescued virion egress indicating that the cytoplasmic terminus of gB is not important for virion egress.