COMPOSITION, ASSEMBLY AND REPLICATION OF RNA VIRUSES

本实验室将对其调控机制进行研究(S) 病毒特异的病毒粒子相关RNA聚合酶的功能 两种负链RNA动物病毒水泡性口炎病毒(VSV) 和甲型流感，具体地说，我们设计了实验来检查 这两种酶的修饰(S)，使它们能够从 将它们的基因组转录成病毒特异性的mRNAs， 甲基化和多腺化，是基因组的不完整拷贝 序列转换为复制模式，从而合成全长(+) 以及在合成过程中组装的基因组的(-)拷贝 核衣壳蛋白形成RNP复合体。 我们目前正在克隆VSV聚合酶(NS)的小亚基，用于 在大肠杆菌和动物细胞中表达，以获得足够数量的 NS，用于分析NS上的磷酸化位点。我们 还将对cdna进行测序，以确定该基因的氨基酸序列 蛋白。如果我们能为NS获得合理的表达水平，我们希望 用纯化的NS蛋白进行体外重组研究 RNPs和L蛋白。 使用单特异性抗血清与两个VSV酶亚基(L1，NS1)进行比较 正在研究这种酶与其RNP模板的相互作用 免疫电子显微镜技术。 因为我们可以抑制流感转录(由于它对宿主的依赖 帽和引物的细胞mRNA)，我们将建立一个在 流感病毒胞核提取物偶联体外复制系统 转录和翻译以类似的方式驱动流感复制 我们的体外VSV复制系统。使用特异性抗体来对抗 流感RNA聚合酶(PB1，PB2PA)我们将确定这三种 亚基参与流感复制和转录。在一个 同样，我们将研究流感非结构蛋白的作用(如果有的话) (NS1)体外复制系统，在其中我们可以选择性地抑制 转录(放线菌D)或复制(放线菌亚胺)来研究酶 修饰(S)以及细胞和/或病毒因素在其中的作用 它。

Our laboratory will study the mechanism(s) involved in the regulation of the functions of the virus-specific, virion-associated RNA polymerases of the two minus-stranded RNA animal viruses, vesicular stomatitis virus (VSV) and influenza A. Specifically we have designed experiments to examine the modification(s) of these two enzymes which allows them to switch from transcription of their genomes into virus-specific mRNAs which are capped, methylated and polyadenylated and are incomplete copies of the genomic sequence to a replicative mode which leads to synthesis of full length (+) and (-) copies of the genome that are assembled during synthesis with nucleocapsid proteins to form RNP complexes. We are presently cloning the small subunit of the VSV polymerase (NS) for expression in E. coli and in animal cells to obtain sufficient amounts of NS with which to carry out analyses of the phosphorylation sites on NS. We will also sequence the cDNA to determine the amino acid sequence of this protein. If we can obtain reasonable levels of expression for NS we hope to use the NS protein for in vitro reconstitution studies with purified RNPs and L protein. Using monospecific antisera versus both VSV enzyme subunits (L1, NS1) we are studying the interaction of this enzyme with its RNP template by immunoelectron microscopic techniques. Since we can inhibit influenza transcription (due to its reliance on host cell mRNA for caps and primers) with actino D, we will establish an in vitro replication system from cell nuclear extracts for flu by coupling transcription and translation to drive flu replication in a manner similar to our in vitro VSV replication system. Using specific antibodies to the flu RNA polymerase (PB1, PB2 PA) we will establish whether all three subunits are involved in flu replication as well as transcription. In a similar manner we will study the role, if any, of flu nonstructural protein (NS1) in vitro replication system in which we can selectively inhibit transcription (actino D) or replication (cycloheximide) to study the enzyme modification(s) involved and the role of cellular and/or virus factors in it.