Analysis of RNA-Protein interactions involved in calicivirus translation and replication

Abstract

The interaction of host-cell nucleic acid-binding proteins with the genomes of positive-stranded RNA viruses is known to play a role in the translation and replication of many viruses. To date, however, the characterisation of similar interactions with the genomes of members of the Caliciviridae family has been limited to in vitro binding analysis. In this study, feline calicivirus (FCV) and murine norovirus (MNV) have been used as model systems to identify and characterise the role of host-cell factors that interact with the viral RNA and RNA structures that regulate virus replication. It was demonstrated that RNA-binding proteins such as polypyrimidine tract-binding protein (PTB), poly(C)-binding proteins (PCBPs) and La protein interact with the extremities of MNV and FCV genomic and subgenomic RNAs. PTB acted as a negative-regulator in FCV translation and is possibly involved in the switch between translation and replication during the late stages of the infection, as PTB is exported from the nucleus to the cytoplasm, where calicivirus replication takes place. Furthermore, using the MNV reverse-genetics system, disruption of 5' end stem-loops reduced infectivity ~15-20 fold, while disruption of an RNA structure that is suspected to be part of the subgenomic RNA synthesis promoter and an RNA structure at the 3' end completely inhibited virus replication. Restoration of infectivity by repair mutations in the subgenomic promoter region and the recovery of viruses that contained repressor mutations within the disrupted structures, in both the subgenomic promoter region and the 3’ end, confirmed a functional role for these RNA secondary structures. Overall this study has yielded new insights into the role of RNA structures and RNA-protein interactions in the calicivirus life cycle.