AMV coat protein-replicase interactions

Positive strand RNA viruses initiate replication through a process where the RNA-dependent RNA polymerase (replicase) binds to exactly the right place on the RNA and starts copying at precisely the correct nucleotide—without the involvement of a primer. How the replicase identifies its binding site and starts copying at a precise position is a remarkable example of the functional specificity of RNA-protein interactions.
The central hypothesis of our work is that the replicase recognizes an organized RNA structure or RNA-protein complex. In many plant viruses, the initiation site for minus strand RNA synthesis includes what is known as a TLS; that is, a transfer RNA-like structure. The TLS fits the criteria of providing a structurally organized domain that could be recognized by the viral replicase.
Our lab works on a different set of viruses—alfalfa mosaic virus (AMV) and ilarviruses—that don’t have a TLS. We are interested in understanding how the replicase recognizes these RNAs and initiates replication at the correct nucleotide.
Through other projects in the laboratory, we have learned how the viral coat protein binds to the initiation site for replication, and how the shapes of the RNA and protein are altered by binding. We hypothesize that the RNA-coat protein interaction facilitates replicase binding, and we are using biochemical methods to examine these binding events. The replicase subunit proteins have been expressed in the laboratory using recombinant baculovirus-infected insect cells. These proteins have been used in far-northwestern probing experiments to suggest that replicase-RNA recognition is facilitated by the viral coat protein. Current experiments are directed at defining the replicase binding site on the viral RNAs and at understanding if the coat protein-RNA interaction forms a binding platform for the replicase.