elF-2 Activity in Vaccinia Virus Infected Cells

痘苗病毒感染细胞中的 eLF-2 活性

• 批准号: 9105451
• 负责人: Rosemary Jagus
• 金额: $ 21.06万
• 依托单位: University of Maryland Biotechnology Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-15 至 1994-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9105451&HistoricalAwards=false
• 关键词: elF; Activity; Vaccinia; Virus; Infected

Eukaryotic initiation factor 2, eIF, is an essential protein synthesis initiation factor that is subject to regulation by reversible phosphorylation. As part of the cellular antiviral defense mechanism, an interferon-induced, dsRNA-dependent protein kinase,eIF-2.-PKds, becomes activated on viral infection and phosphorylates the alpha subunit eIF-2. This results in a limitation of functional eIF-2 available for protein synthesis with a concomitant reduction of protein synthesis. A number of animal viruses, including vaccinia virus, have evolved mechanisms to inhibit the activation of eIF-2.-PKds in interferon-treated cells. The ability of vaccinia virus to grow in interferon-treated cells stems in part from the production of a vaccinia early gene product, termed specific kinase inhibitory factor, SKIF, that prevents activation of eIF-2.-PKds. In addition vaccinia virus encodes a gene which has homology with the alpha subunit of eIF-2 and encodes a 10 kDa protein. Although the function of the eIF-2 alpha-like gene is not known, an eIF-2 alpha-like peptide of approximately 10 kDa accumulates early in vaccinia virus infection. The first aim of this proposal is to monitor the effect of vaccinia virus infection on the levels and activation state of elF-2.-PKds, as well as the phosphorylation state of elF-2. in vaccinia virus infected Hela cells. The reagents necessary to do this, antibody to elF-2.-PKds and elF-2. are available. The second aim of ths ptoposal is to investigate the function of pK2 and to determine whether its product has elF-2>.-like activity and whether it is involved in the ability of vaccinia virus to grow in interferon- treated cell. The K2 gene has been cloned into the expression vector, pTM1, which will enable us to synthesize radiolabelled PK2 in vitro, as well to transfect the gene into mammalian cells. The gene will be transferred to a bacterial expression vector to allow the production of milligram quantities of pK2 for biochemical analyses. The properties of the vaccinia encoded elF-2.-like gene product will be compared with those of cellular elF-2. with reference to its guanine nucleotide binding properties, its ability to enter preinitiation complexes, and its ability to function as a substrate for elF-2.-PKds.

真核起始因子2（eIF）是一种重要的蛋白质 合成起始因子，受 可逆磷酸化 作为细胞抗病毒药的一部分 防御机制，一种干扰素诱导的dsRNA依赖蛋白 激酶，eIF-2。PKds在病毒感染时被激活， 磷酸化α亚基eIF-2。 这导致 限制可用于蛋白质合成的功能性eIF-2， 蛋白质合成的减少。 许多动物 包括牛痘病毒在内的病毒已经进化出了 抑制eIF-2的激活。干扰素处理的细胞中的PKd。 牛痘病毒在干扰素处理的细胞中生长的能力 部分源于牛痘早期基因产物的产生， 称为特异性激酶抑制因子，SKIF， 激活eIF-2。PKD 此外，牛痘病毒编码 与eIF-2的α亚基具有同源性并编码 10 kDa蛋白质。 虽然eIF-2 α样蛋白的功能 基因是未知的，eIF-2 α样肽大约 10 kDa在牛痘病毒感染早期积累。 这项建议的第一个目的，是监察牛痘的效果 病毒感染对eIF-2的水平和激活状态的影响。PKD， 以及eIF-2的磷酸化状态。在痘苗病毒 感染的Hela细胞。 所需的试剂，抗体 到elF-2 PKD和elF-2。都是可用的。 第二个目标 目的是研究pK 2的功能，并确定 其产品是否具有elF-2>。比如活动， 与牛痘病毒在干扰素中生长的能力有关， 处理过的细胞 K2基因已被克隆到表达载体中， 载体pTM 1，这将使我们能够合成放射性标记的PK 2 在体外，以及将该基因导入哺乳动物细胞。 的 将基因转移到细菌表达载体中， 用于生物化学的毫克量的pK 2的产生 分析。 牛痘编码的eIF-2的性质。样基因 将产物与细胞eIF-2的那些进行比较。与 参考其鸟嘌呤核苷酸结合特性，其 进入preinitiation复合体的能力， 作为elF-2的底物。PKD