Replication protein of tobacco mosaic virus cotranslationally binds the 5' untranslated region of genomic RNA to enable viral replication.
Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5'-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5'-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided.
Plant-Microbe Interactions Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Kazue
K
Kawamura-Nagaya
Kazuhiro
K
Ishibashi
Ying-Ping
YP
Huang
Shuhei
S
Miyashita
Masayuki
M
Ishikawa
D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D002850:Chromatography, Gel; D016679:Genome, Viral / Q000235:genetics*; D008836:Micrococcal Nuclease / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
D020121:5' Untranslated Regions; D012367:RNA, Viral; D014764:Viral Proteins; D012260:Ribonucleases; D008836:Micrococcal Nuclease