The importance of the UTR panhandle structure of VHS virus for replication and virulence

It has been difficult to pinpoint virulence markers in VHS virus and the understanding of the replication mechanisms of the virus is also scarce. Studies in model viruses like vesicular stomatitis virus have shown that the 3-terminus is complementary to t he 5-terminal end and the complementary of genomic termini allows formation of a panhandle structure. Previous studies have shown that the extent of complementarity of the 3 and 5 end modulates a balance between transcription and replication. It has also been shown that mismatches within the 3 and 5 ends seem to play a role in balancing the transcriptional promoter activity and replicative activity. Furthermore, it has shown that transcription of viral mRNA and replication is initiated at different sites for vesicular stomatitis virus. This will have implication for replication capacity and potentially virulence of the virus. These mechanisms have been studied in the Rhabdoviruses but are unknown for the Novirhabdoviruses. In this proposal we hypothesi ze that there are specific residues of the internal loop of double-stranded panhandle structure that serve as promoter with specific determinants for RdRp binding. From preliminary studies using reverse genetics we have point mutated residues of the inter nal loop of 3?end terminal of VHSV genotype IVa strain and used single-sided specific first cDNA synthesis to assess viral transcription from viral RNA to mRNA or cRNA, and subsequently viral replication from cRNA to viral RNA. There are strong indication s that the panhandle structure and specific residues are important for replication of VHS virus. We will study their importance by use of reverse genetics, perform in vitro studies and perform preliminary in vivo experiments using zebrafish challenge mode ls.