RNA replicons function as genomes for a large number of animal, human, plant and insect viruses. Despite the increased vulnerability of RNA for nucleolytic breakdown, RNA viruses continue to overpopulate the virus world. High mutation rates and RNA-RNA recombination are believed to play a critical role in RNA genome evolution, persistence and pathogenesis. Arboviruses, the largest group of animal RNA viruses, because of their wide tissue and host specificity, are forced to evolve faster than other viruses. Sindbis virus (SIN), the subject of this proposal, is a mosquito-transmitted human alphavirus. Many human alphaviruses including Western equine encephalitis (WEE) have evolved by genetic recombination between SIN and Eastern equine encephalitis virus (EEE). Furthermore, the emerging use of alphavirus RNA replicons in gene therapeutics and vaccines warrants a detailed understanding of the biology of RNA replicons. The long term goal of this project is to understand the mechanism of alphavirus RNA repair and recombination in infected host cells. Infectious cDNA clones of SIN are engineered to express a battery of replicative and nonreplicative RNA precursors. Both mammalian and mosquito cells are transfected with these engineered RNAs, and the ability of the viral polymerase to repair and generate infectious virus from these host cells are characterized. There are three specific aims: (1) To study the role of nucleotide homology between template RNAs in homologous recombination; (2) To study the role of template-template interaction, RNA structures and cryptic promoter-like elements in the generation of nonhomologous SIN recombinants; and (3) To identify the amino acid changes in virus polymerase proteins which render SIN RNAs recombination-defective.
|James, Frederick D; Hietala, Katie A; Eldar, Dganit et al. (2007) Efficient replication, and evolution of Sindbis virus genomes with non-canonical 3'A/U-rich elements (NC3ARE) in neonatal mice. Virus Genes 35:651-62|