title: Cleavage site mutations in the encephalomyocarditis virus P3 region lethally abrogate the normal processing cascade.
abstract: Site-specific mutations within the proteinase 3C-dependent P3 region cleavage sequences of encephalomyocarditis virus have been constructed. The mutations altered the normal QG cleavage site dipeptide pairs of the 2C/3A, 3A/3B, 3B/3C, and 3C/3D junctions into QV, QC, QF, QY, and RG sequences. When translated in vitro in the context of full-length viral polyproteins, all mutations blocked endogenous 3C-mediated processing at their engineered sites and produced stable forms of the expected viral P3 precursors that were also resistant to cleavage by exogenously added recombinant 3C. Relative to wild-type viral sequences, each mutant form of P3 had a somewhat different ability to mediate overall polyprotein processing. Mutations at the 2C/3A, 3A/3B, and 3B/3C sites, for example, were generally less impaired than 3C/3D mutations, when the cleavage reactions were quantitated with cotranslated L-P1-2A precursors. A notable exception was mutant 3B3C(QG-->RG), which proved far less active than sibling mutants 3B3C(QG-->QF) and 3B3C(QG-->QV), a finding that possibly implicates this segment in the proper folding of an active 3C. When transfected into HeLa cells, all mutant sequences were lethal, presumably because of the reduced L-P1-2A processing levels or reduced RNA synthesis capacity. However, when specifically tested for the latter activity, all mutations except those at the 3C/3D cleavage site were indeed able to initiate and perpetuate viral RNA replication in transfected cells, albeit to RNA accumulation levels lower than those produced by wild-type sequences. The transfection effects could be mimicked with cell-free synthesized proteins, in that translation samples containing locked 3CD polymerase precursors were catalytically inactive in poly(A)-oligo(U)-dependent assays, while all other mutant processing samples initiated detectable RNA synthesis. Surprisingly, not only did the 3B/3C mutant sequences prove capable of directing RNA synthesis, but the viral RNA thus synthesized could be immunolabeled and precipitated with 3C-specific monoclonal antibody reagents, indicating an unexpected covalent attachment of the proteinase to the RNA product whenever this cleavage site was blocked.
authors.affiliation: Institute for Molecular Virology, University of Wisconsin-Madison 53706, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Hall
authors.forename: A C
authors.initials: AC
authors.lastname: Palmenberg
mesh_terms: D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D018076:DNA, Complementary; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000502:physiology*; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D011817:Rabbits; D015183:Restriction Mapping; D012156:Reticulocytes; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis*; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases