Grant: Discovery Grants Program - Individual

Like other viruses, avian reoviruses (ARVs) utilize the host's cell metabolic system for their own survival and replication. This process is almost entirely dependent on temporally and spatially regulated ARV-host protein-protein interactions. As viruses evolve, amino acid substitutions in functional domains of viral proteins affect viral protein folding, stability, and protein function by altering the interaction of viral proteins with host macromolecules. ARVs contain 10 segments of double stranded RNA genome. Earlier, our studies demonstrated that ARVs rapidly evolve and adapt through a combination of accumulation of spontaneous mutations, genetic re-assortment and recombination (currently, six genotyping cluster groups of ARV have been identified). Our Next Generation Sequencing (NGS) experiments on several emerging ARV variants revealed a significant sequence divergence at the whole genome level, which resulted in differences in the degree of virulence between the different isolates. Therefore, we believe that natural non-synonymous single nucleotide polymorphisms (nsSNPs; nucleotide mutations which cause changes in the amino acid sequence of viral proteins) alter the structure and conformation of the functional domains of viral proteins and the dynamics of ARV-host interplay. Since the highest rate of nsSNPs was identified in genome segment S1 of the ARV isolates in our comparative individual RNA gene segment analysis, the aim of this project is to study the effects of nsSNPs in genome segment S1 (i.e., encodes s-C, p10 and p17 proteins) in virus protein function during virus replication. This goal will be achieved by 1) analysis of differentially expressed genes in avian cell lines transfected in-vitro with plasmid DNA constructs which express variants of s-C, p10 and p17 proteins by mRNA sequencing using NGS technology; 2) identification of important cellular protein-networks which interact with natural variants of s-C, p10 and p17 proteins; 3) mapping of distinct functional domains among natural variants of s-C, p10 and p17 proteins (by sequential and/or internal deletions); and 4) developing a plasmid-based ARV reverse genetics system. The results of this study will a) lead to a better understanding of the impact of evolutionary divergence of ARV S1 genomic segment on s-C, p10 and p17 proteins function as well as their effects on cellular metabolism; b) give insight to conserved functional domains of the S1 gene encoded proteins which maintain essential functions for progeny virus production; c) advance the knowledge on the molecular biology of ARV and other viruses in the orthoreovirus genus; and d) help in developing effective vaccines, diagnostic systems and novel antiviral targets.