Towards a unified model for initiation of the first step of picornavirus genome replication

Viruses of the Picornaviridae family are positive-stranded RNA viruses with important animal and human pathogens. Poliovirus (PV) is a prototype virus of this family. Even though detailed models describing the mechanism of replication of this virus appear in virology textbooks, the studies detailed in this thesis have provided compelling evidence that these current models need to be revisited.;VPg is a 22 amino acid peptide found covalently linked to the 5' ends of both minus and plus strands during PV genome replication. This peptide is used by the RNA-dependent RNA polymerase (RdRP) as a primer during RNA synthesis. The primer is first "activated" in the middle of the genome and then requires transfer to the 3' end where it is then extended to produce a full-length copy of the genome. This activation step involves the covalent attachment of two uridine monophosphates to the hydroxyl of a tyrosine in VPg. Using an in vitro biochemical assay (the VPg uridylylation assay) and other well established cell culture assays designed to evaluate RNA synthesis and virus multiplication, I have demonstrated important aspects related to the initiation step which should help rewrite several dogmas regarding PV genome replication.;I demonstrate the existence of a novel interaction between key replication proteins essential for this initiation step. The data presented also provide a possible mechanism for the transfer of the "activated" protein primer from the center of the genome to the 3' end. They question the biological significance of interactions between RdRP molecules that were thought to be essential for virus multiplication.;I present evidence that shows that a precursor form of the VPg peptide is more efficient in the in vitro uridylylation assay. The production of precursor-linked genomes in cell culture experiments and evidence of a dual protein processing cascade for proteins directly involved with genome replication have provided valuable insight into the protein players involved in this process. These data challenge the dogma that the processed forms of the replication proteins which accumulate within infected cells are, indeed, the same forms of the proteins required for establishment of the replication complex.