The Replicative Complex of Paramyxoviruses: Structure and Function

Abstract

Publisher Summary In paramyxoviruses, the nucleocapsid is not only a structural component used to enwrap the genome but also a functional entity required for virus replication. Therefore, all protein components of the paramyxoviral nucleocapsid are involved in manifold activities. Not only is the three-dimensional structure of each entire protein subunit important in building up the viral replicative complex but also the specific protein-RNA and proteinprotein interactions are essential for bringing the components into the right contact with each other and to regulate enzymatic activities. This chapter discusses the methods to monitor the influence of defined nucleotide or amino acid exchanges on the assembly of nucleocapsids, transcription and replication of the viral RNA, and rescue of virus particles—that is, to study the regulation of paramyxoviruses replication during the whole viral life cycle. To explore domains involved in protein-protein interactions, viral wild-type proteins or specifically modified variants expressed in prokaryotic or eukaryotic cells were investigated using a variety of techniques such as protein-protein overlay assays, immunocoprecipitation, cosedimentation, and the two-hybrid system. Furthermore, as a prerequisite for functional investigations of the viral replicative complex— in vitro and in vivo —RNA transcription and/or replication assays were developed either by assembling nucleocapsids out of several protein-RNA and protein-protein complexes or by expressing all essential components together in eukaryotic cells, thus forming a functional viral nucleocapsid.