STUDIES ON THE TRANSLATION AND REPLICATION OF ENCEPHALOMYOCARDITIS VIRUS

Abstract

The mechanism of translation and replication of picornaviruses was investigated using Encephalomyocarditis (EMC) virus as a model system. In the first part of this work, antisense oligonucleotides designed to hybridize to various regions within the viral RNA were used as tools to map the functionally important regions required for translation initiation in vitro. A significant degree of translation inhibition was observed with oligonucleotides complimentary to the 5 'non-translated region and AUG initiation codon. However in the case of the coding-region -specific oligonucleotide, no inhibition was observed unless the oligonucleotide:RNA hybrid was digested with RNase H prior to translation in vitro, Taken together, the data demonstrate the efficient inhibition of translation of EMC virus RNA in vitro using short antisense oligonucleotides complementary to the 5' non-translated region. The second part of this project involved the study of EMC virus RNA-dependent RNA polymerase ( 3Dpol) , the enzyme involved in the replication of these viruses. As a first step, the viral cDNA encoding the putative RNA-dependent RNA polymerase was expressed in E.coli using the glutathione S-tranferase expression system which enabled easy purification of the recombinant enzyme. The purified recombinant EMC virus 3Dpol has a molecular mass of 52kDa and is recognized by polyclonal antisera raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme exhibits poly(U) and RNA polymerase activities analogous to that reported for the poliovirus enzyme. The enzyme was characterized with respect to its sensitivity towards known inhibitors of polymerases and nucleotide analogues. The studies show that the enzyme is distinct from the reverse transcriptases, bacterial DNA-dependent RNA polymerase and eukaryotic viral DNA polymerases that are a-like. The enzyme is sensitive to N-ethylmaleimide and cerulenin, an inhibitor of phospholipid biosynthesis. In addition, nucleotide analogues with modified 3'-OH groups that act as chain terminators inhibit enzyme activity. An extensive analysis of the structure-function relationship of EMC virus 3Dpol was carried out by site-directed mutagenesis and the activities of the mutant polymerases assayed using an in vitro poly(U) polymerase assay system. Seventeen amino acid substitutions were carried out based on sequence alignments of RNA-dependent RNA polymerases of viral origin that identified highly conserved amino acid residues within motifs. Thirteen out of the seventeen conservative substitutions within the four most conserved motifs' reduced the RNA polymerase activity of the mutants to 0-30% of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol, The positions of these substitutions were compared with the known three-dimensional structure of E.coli DNA polymerase I (Klenow). Remarkably, five of the ten mutations in EMC virus 3JJP01 which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in Klenow which together perform important functions in substrate binding and DNA polymerization. The results reveal that a basic structural framework is conserved in the most distantly related classes of nucleic acid polymerases, and demonstrates the validity of modelling the active site of RNA -dependent RNA polymerases on the known structure of a DNA polymerase.