Chapter 5. In vivo analysis of the decay of transcripts generated by cytoplasmic RNA viruses

The field of RNA decay has grown extensively over the last few years and numerous decay pathways have been identified and characterized. This is a truly powerful machinery for both regulation and quality control of gene expression. It is very likely that the transcripts of RNA viruses must successfully confront this arsenal of enzymes and RNA binding factors in order to establish a productive infection. This interface is an understudied branch of virology that needs to be explored if we are to fully comprehend the molecular biology of virus-cell interactions. Research in this area has the potential to increase our understanding of the fundamentals of both mRNA stability and viral biology, perhaps leading to novel antiviral approaches. This chapter discusses methods for examining the half-lives of viral RNAs during natural infection, including purification of the viral transcripts and subsequent analysis of both deadenylation and decay. Additionally, a hybrid selection protocol for identifying viral-specific small RNAs that are generated during infection by the RNAi branch of the cellular RNA decay machinery is described.