title: A C-terminal, cysteine-rich site in poliovirus 2C(ATPase) is required for morphogenesis.
abstract: The morphogenesis of viruses belonging to the genus Enterovirus in the family Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2C(ATPase) gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. The polypeptide 2C(ATPase) is a highly conserved non-structural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C terminus of the polypeptide that is required for morphogenesis. The aim of the current project was to search for additional functional sites near the C terminus of the 2C(ATPase) polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding two lethal, one temperature-sensitive and one quasi-infectious mutants. All four mutants exhibited normal protein translation in vitro and three of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudo-reversion at the original site (A286D), but some also contained one additional mutation: A138V or M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation and possibly also an uncoating defect at 37 °C. Variants of this mutant revealed suppressor mutations at three different sites in the 2C(ATPase) polypeptide: A138V, M293V and K295R. We concluded that the cysteine-rich site near the C terminus of 2C(ATPase) is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain.
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Chunling
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Hsin-Chieh
authors.initials: HC
authors.lastname: Ma
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Eckard
authors.initials: E
authors.lastname: Wimmer
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Jiang
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Aniko V
authors.initials: AV
authors.lastname: Paul
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D009154:Mutation; D010641:Phenotype; D017955:Poliovirus / Q000235:genetics / Q000254:growth & development* / Q000502:physiology*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C076023:2C protein, viral; D000251:Adenosine Triphosphatases