Molecular mechanism of homologous viral interference

Vesicular stomatitis virus (VSV) is comprised of two serotypes, Indiana serotype (VSV_IND) and New Jersey serotype (VSV_NJ). VSV _NJ is further divided into two subtypes, Hazelhurst (VSV_NJ-Haz ) and Ogden (VSV_NJ-Ogd). Defective interfering (DI) particles are generated from undiluted high multiplicity passages of the standard virus. The DI particles from VSV_IND and VSV_NJ interfere with the replication of the standard virus, inhibiting standard virus genomic RNA and mRNA synthesis. VSV_IND DI particles interfere with the replication of VSV_IND (homotypic interference), as well as VSV_NJ (heterotypic interference). In contrast, VSV_NJ DI particles only interfere with the replication of the homotypic standard virus. To better understand the molecular mechanism of homotypic and heterotypic viral interference, I examined the replication and assembly of panhandle type DI particle genomes during concurrent expression of VSV proteins from both serotypes.; For the analyses of replication and assembly of DI particles by a VSV reverse genetics system, cDNA plasmids of all 5 VSV genes from both serotypes were required. However, a cDNA plasmid of the VSV_NJ L gene, which encodes VSV RNA polymerase, was not available. Therefore, I cloned cDNAs of VSV_NJ-Haz L gene (pHL) and VSVNJ-Ogd L gene (pOL). The cloned pHL and pOL expressed functional RNA dependent RNA polymerase, as determined by transcription and replication analyses.; Replication and assembly analyses of VSV DI particle genomes were carried out using plasmids encoding chimeric DI particle genomes carrying bacteriophage sequences (DI-λ RNA). DI-λ RNA contains various lengths of 3 ′ and 5′ genomic termini of VSV DI particles and bacteriophage λ sequences, replacing the internal region of the DI particle genome. Differing efficiencies of replication and assembly using homotypic and heterotypic proteins was demonstrated between VSV_IND and VSV_NJ DI particles, as well as between VSV_NJ DI particles containing different lengths of genomic end sequences. Although genomic RNAs of VS_VNJ DI particles replicated using VSV_IND N, P, and L proteins, the efficiency was considerably lower, indicating the presence of VSV_IND specific sequences in the promoter of the VSV_IND DI particle genome. I have also demonstrated that the length of the type-specific promoter region in the VSV_NJ DI particle genome is longer than 50 nucleotides. The examination of the assembly of DI-λ particles in the presence of homotypic and heterotypic proteins demonstrated that the terminal genomic sequences might have an essential role in the assembly of VSV.; In addition, I have defined the VSV_IND specific promoter sequences at the genomic termini of VSV_IND DI particle. The sequences were located between nucleotides 26 and 45 from the 3′ and 5′ ends of the DI particle genome.; Since the panhandle type DI particles are lacking functional open reading frames to encode VSV proteins, utilization of homotypic and heterotypic proteins from the standard virus may be crucial in homotypic and heterotypic viral interference. Therefore, the results presented in this thesis attempt to explain the possible mechanisms of homotypic and heterotypic viral interference.