GENETICS AND FUNCTIONS OF KSHV GLYCOPROTEINS GM AND GN

KSHV 糖蛋白 GM 和 GN 的遗传学和功能

• 批准号: 7719996
• 负责人: OSWALD D'AUVERGNE
• 金额: $ 12.41万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7719996
• 关键词: Amino Acids; B-Cell Lymphomas; Bacterial Artificial Chromosomes; Body cavities; Cell Line; Cell fusion; Cells; Codon Nucleotides; Computer Retrieval of Information on Scientific Projects Database; Escherichia coli; Facility Construction Funding Category; Funding; Genes; Genome; Glycoproteins; Goals; Grant; Herpesviridae; Human Herpesvirus 8; Institution; Kaposi Sarcoma; Life Cycle Stages; Lymphoma; Morphogenesis; Multicentric Angiofollicular Lymphoid Hyperplasia; Oncogenic Viruses; Pathogenesis; Plasmids; Play; Research; Research Personnel; Resources; Role; Simplexvirus; Source; Specific qualifier value; Structure; Transfection; United States National Institutes of Health; Viral; Viral Genome; Virion; Virus; Virus Activation; Work; base; body cavity; effusion; lytic replication; mutant

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 卡波西肉瘤相关疱疹病毒 (KSHV) 或人疱疹病毒 8 型是一种嗜淋巴细胞致癌病毒，与卡波西肉瘤的发病机制有关。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卡斯尔曼病。与其他疱疹病毒类似，KSHV 编码许多糖蛋白，这些糖蛋白在病毒颗粒形态发生和感染性中发挥重要作用。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，最初重点关注糖蛋白 gM 和 gB。已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。通过将 gM 基因插入大肠杆菌 KSHV-bac36 基因组中，构建 gM 无效 KSHV 突变病毒。 将 KSHV gM 缺失的病毒基因组转染到 293 细胞中产生了病毒粒子，这些病毒粒子能够从转染的细胞中逸出并感染其他细胞，表明 gM 对于病毒粒子的逸出和感染性并不重要。众所周知，KSHV gB 在病毒颗粒排出中很重要。我们对 gB 的研究重点是 gB 细胞质末端在病毒诱导的细胞融合和病毒颗粒排出中的作用。构建了特异 gB 羧基末端缺失的突变 KSHV 病毒。 gB 中 25 个氨基酸的缺失显着增强了病毒诱导的细胞融合。为了使用BCBL-1细胞系描绘参与病毒颗粒排出的gB功能域，BCBL-1细胞系在诱导病毒裂解性复制时产生大量病毒，使用了gB特异性的siRNA。 将 siRNA 转染到 BCBL-1 细胞中显着减少了 BCBL-1 细胞中的病毒颗粒排出。 用密码子优化的 gB 转染抗 gB siRNA 抑制的 BCBL-1 细胞可挽救病毒粒子的流出。类似地，用表达 gB 截短的质粒转染可挽救病毒粒子的流出，表明 gB 的细胞质末端对于病毒粒子的流出并不重要。