An ideal tool to study biosynthesis pathways in mammalian cells is a virus which interferes with these processes. Poliovirus is a positive-strand cytoplasmic RNA virus that undergoes extensive interactions with its host cell, including the inhibition of cellular transcription and translation. The discovery that a cDNA clone of the viral genome gives rise to infectious virus upon introduction into mammalian cells, created the opportunity to construct defined viral mutants by site-directed mutagenesis and to study their biological properties.
The aim of this proposal is to apply genetics and biochemistry to identify functions of a viral RNA genome and its interaction with the host cell. Using a temperature-sensitive (ts) poliovirus mutant, 3NC202, which bears an eight nucleotide insertion in the 3' noncoding region and is defective in the initiation of minus-strand RNA, we will identify structural and functional elements in the RNA molecule which control the amplification of negative-strand RNA. Specifically, we are going to analyze the structure of the mutant RNA using chemical probes and reverse transcriptase. In addition, we will search for viral trans-acting factors which might interact with the 3' untranslated region. Therefore, revertants of the ts-mutant will be isolated, and the altered protein factor tested for its ability to bind to the viral """"""""ts-RNA"""""""" molecule. In addition, we will study the biological properties of a small plaque mutant, 2B201, mapping in the gene encoding 2B, a protein with unknown function. Genetics (analysis of revertants) and biochemical approaches (isolation and in vitro assays of 2B) will be applied to gain understanding of the role of the 2B polypeptide in the viral life cycle. We will also investigate why the cellular mRNA encoding the glucose regulated protein (GRP80) is translated in mutant-infected cells at a time when the translation of other host mRNA molecules is inhibited. Translatability of hybrid-selected GRP80 mRNA in infected and uninfected cellular extracts will be analyzed. We will construct hybrid genes between GRP80 cDNA sequences and a test gene and examine translation of in vitro made transcripts, to identify sequences which are involved in cap- independent translation. The health relatedness of this project lies in its contribution to the understanding of how animal RNA viruses interact with their host cells. Poliovirus is one of the best studied animal viruses because its genome organization is known and its three dimensional structure has been solved. The recent sequencing of other RNA virus genomes has revealed great homology between the members of the picornaviridae family. Thus, knowledge of the structure and function of the poliovirus genome might be applicable to the genomes of other medically important RNA viruses such as foot and mouth disease virus and human rhinovirus, responsible for the common cold.
