elF-2 Activity in Vaccinia Virus Infected Cells

真核起始因子2 （Eukaryotic initiation factor 2, eIF）是一种必需的蛋白质合成起始因子，受可逆磷酸化调控。作为细胞抗病毒防御机制的一部分，干扰素诱导的dsrna依赖性蛋白激酶eIF-2。-PKds，在病毒感染时被激活并磷酸化α亚基eIF-2。这就限制了用于蛋白质合成的功能性eIF-2，同时也减少了蛋白质合成。许多动物病毒，包括牛痘病毒，已经进化出抑制eIF-2激活的机制。-干扰素处理细胞中的pkds。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），它可以阻止eif -2 - pkds的激活。此外，牛痘病毒编码一个与eIF-2 α亚基同源的基因，并编码一个10 kDa的蛋白。虽然eIF-2 α样基因的功能尚不清楚，但约10 kDa的eIF-2 α样肽在牛痘病毒感染早期积累。本研究的第一个目的是监测牛痘病毒感染对elF-2水平和激活状态的影响。-PKds，以及elF-2的磷酸化状态。牛痘病毒感染海拉细胞需要的试剂，elF-2抗体。- pkd和精灵2。是可用的。本提案的第二个目的是研究pK2的功能，并确定其产物是否具有elF-2>。样活性以及它是否参与牛痘病毒在干扰素处理细胞中生长的能力。K2基因已被克隆到表达载体pTM1中，这将使我们能够在体外合成放射性标记的PK2，并将该基因转染到哺乳动物细胞中。该基因将被转移到细菌表达载体上，以便产生毫克数量的pK2进行生化分析。牛痘编码elF-2的性质。样基因产物将与细胞elF-2的产物进行比较。考虑到其鸟嘌呤核苷酸结合特性，进入起始前复合物的能力，以及作为elf -2 - pkds底物的能力。

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.