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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.

SUBMITTER: Ogden KM

PROVIDER: S-EPMC3067766 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication.

Ogden Kristen M KM Ramanathan Harish N HN Patton John T JT

Journal of virology 20101208 5

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 s ...[more]

PMID: 21147920

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Project description:Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny.Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.

Dataset Information

Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication.

Publications

Residues of the rotavirus RNA-dependent RNA polymerase template entry tunnel that mediate RNA recognition and genome replication.

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