title: Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication.
abstract: To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (+RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3' end of rotavirus +RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral +RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with +RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of +RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates.
authors.affiliation: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Ogden
authors.forename: Harish N
authors.initials: HN
authors.lastname: Ramanathan
authors.forename: John T
authors.initials: JT
authors.lastname: Patton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004261:DNA Replication; D016679:Genome, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012401:Rotavirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D013698:Templates, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase