Sharma Amit, Boris-Lawrie Kathleen
Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio, USA.
Methods Enzymol. 2012;511:405-35. doi: 10.1016/B978-0-12-396546-2.00019-X.
RNA helicases are encoded by all eukaryotic and prokaryotic cells and a minority of viruses. Activity of RNA helicases is necessary for all steps in the expression of cells and viruses and the host innate response to virus infection. Their vast functional repertoire is attributable to the core ATP-dependent helicase domain in conjunction with flanking domains that are interchangeable and engage viral and cellular cofactors. Here, we address the important issue of host RNA helicases that are necessary for replication of a virus. This chapter covers approaches to identification and characterization of candidate helicases and methods to define the biochemical and biophysical parameters of specificity and functional activity of the enzymes. We discuss the context of cellular RNA helicase activity and virion-associated RNA helicases. The methodology and choice of controls fosters the assessment of the virologic scope of RNA helicases across divergent cell lineages and viral replication cycles.
RNA解旋酶由所有真核细胞、原核细胞以及少数病毒编码。RNA解旋酶的活性对于细胞和病毒表达的所有步骤以及宿主对病毒感染的固有反应都是必需的。它们广泛的功能归因于核心ATP依赖性解旋酶结构域以及与之相连的可互换结构域,这些结构域可与病毒和细胞辅因子结合。在这里,我们探讨了病毒复制所必需的宿主RNA解旋酶这一重要问题。本章涵盖了鉴定和表征候选解旋酶的方法,以及定义这些酶特异性和功能活性的生化和生物物理参数的方法。我们讨论了细胞RNA解旋酶活性和病毒体相关RNA解旋酶的情况。方法学和对照的选择有助于评估RNA解旋酶在不同细胞谱系和病毒复制周期中的病毒学作用范围。