Balachandran Siddharth, Barber Glen N
University of Miami School of Medicine, FL, USA.
Methods Mol Biol. 2007;383:277-301. doi: 10.1007/978-1-59745-335-6_18.
The mammalian innate immune system provides a first line of defense against microbial pathogens and also serves to activate an antigen specific acquired immune program. Key components of innate immunity are the interferons (IFNs), a family of related cytokines with potent antimicrobial and immuno-modulatory activities. The IFNs exert their effects through the induction of numerous genes, one of which is the double-stranded RNA-dependent protein kinase (PKR), a pivotal antiviral protein found in most human cells. Following activation by double stranded (ds) RNAs produced during viral replication, PKR phosphorylates the alpha-subunit of eukaryotic translation initiation factor (eIF) 2, causing a severe inhibititon of cellular and viral protein synthesis. Phosphorylation of eIF2alpha and consequent inhibition of protein synthesis is a major cell growth checkpoint utilized by at least three other kinases, in addition to PKR, following exposure to such cellular stresses as amino acid deprivation and the presence of misfolded proteins in the endoplasmic reticulum. Indeed, it has been demonstrated that disruption of the eIF2alpha checkpoint can lead to the transformation of immortalized rodent and human cells, plausibly by increasing the protein synthesis rates of proto-oncogenes. Further, it has been shown that disregulation of the eIF2alpha checkpoint and consequent permissiveness to virus infection may be a common occurrence in tumorigenic mammalian cell lines. These findings have been exploited to develop potent oncolytic RNA viruses that can selectively replicate in and destroy a variety of neoplasias in vitro and in vivo. In this chapter, we describe some of the techniques commonly used in our laboratory to examine PKR activity and eIF2 regulation. Protocols for the generation and use of recombinant vesicular stomatitis virus variants are also described.
哺乳动物的固有免疫系统是抵御微生物病原体的第一道防线，同时也有助于激活抗原特异性获得性免疫程序。固有免疫的关键组成部分是干扰素（IFN），这是一类相关的细胞因子，具有强大的抗菌和免疫调节活性。干扰素通过诱导众多基因发挥作用，其中之一是双链RNA依赖性蛋白激酶（PKR），它是大多数人类细胞中一种关键的抗病毒蛋白。在病毒复制过程中产生的双链（ds）RNA激活后，PKR会使真核翻译起始因子（eIF）2的α亚基磷酸化，从而严重抑制细胞和病毒蛋白的合成。eIF2α的磷酸化以及随之而来的蛋白质合成抑制是至少三种其他激酶利用的主要细胞生长检查点，除了PKR之外，在暴露于诸如氨基酸剥夺和内质网中错误折叠蛋白的存在等细胞应激之后也是如此。事实上，已经证明破坏eIF2α检查点可能导致永生化啮齿动物和人类细胞的转化，可能是通过提高原癌基因的蛋白质合成速率。此外，已经表明eIF2α检查点的失调以及随之而来的对病毒感染的允许性在致瘤性哺乳动物细胞系中可能是常见的现象。这些发现已被用于开发强大的溶瘤RNA病毒，其可以在体外和体内选择性地在各种肿瘤中复制并破坏它们。在本章中，我们描述了我们实验室中常用的一些检测PKR活性和eIF2调节的技术。还描述了重组水疱性口炎病毒变体的产生和使用方案。
