title: Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes.
abstract: We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.
authors.affiliation: Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Med. Sci. B240, Irvine, CA 92697, USA.
authors.forename: Jo Ellen
authors.initials: JE
authors.lastname: Brunner
authors.forename: Joseph H C
authors.initials: JH
authors.lastname: Nguyen
authors.forename: Holger H
authors.initials: HH
authors.lastname: Roehl
authors.forename: Tri V
authors.initials: TV
authors.lastname: Ho
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Swiderek
authors.forename: Bert L
authors.initials: BL
authors.lastname: Semler
mesh_terms: D000086042:3C Viral Proteases; D019943:Amino Acid Substitution; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C / Q000378:metabolism*; D006801:Humans; D017955:Poliovirus / Q000502:physiology*; D011485:Protein Binding; D012367:RNA, Viral / Q000096:biosynthesis*; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
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