Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity

Gomatos, Peter J.; Stamatos, Nicholas M.; Sarkar, Nurul H.

doi:10.1128/jvi.36.2.556-565.1980

Cited by 21 publications

(17 citation statements)

References 24 publications

(22 reference statements)

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“…Despite this limitation, analysis of the BTV protein NS2 (41 kDa) and the reovirus protein NS (41 kDa) suggest that they may be the homologs of rotavirus NSP2. All of these proteins have been implicated in viral genome replication and/or packaging (Gillian and Nibert, 1998;Huismans and Joklik, 1976;Gomatos et al, 1980Gomatos et al, , 1981Thomas et al, 1990).…”

Section: Homologs Of Nsp2 and Nsp5 For Reovirus And Bluetongue Virus mentioning

confidence: 99%

“…Like rotavirus NSP2, BTV NS2 and reovirus NS are expressed in high levels in the infected cell where they localize to VIBs (Brookes et al, 1993;Thomas et al, 1990, Becker et al, 2001. The proteins are found associated with viral mRNA during replication (Antczak and Joklik, 1992;Huismans et al, 1987;Helmberger-Jones and Patton, 1986) and, as with rotavirus NSP2, NS was also found associated with particles that had replicase activity (Gomatos et al, 1980). The three proteins, rotavirus NSP2, BTV NS2 and reovirus NS, share two common and distinctive features that may be critical for their role in genome replication and/or packaging.…”

Section: Homologs Of Nsp2 and Nsp5 For Reovirus And Bluetongue Virus mentioning

confidence: 99%

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Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Taraporewala

Patton

2004

Virus Research

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Rotaviruses, members of family Reoviridae, are a major cause of acute gastroenteritis of infants and young children. The rotavirus genome consists of 11 segments of double-stranded (ds)RNA and the virion is an icosahedron composed of multiple layers of protein. The virion core is formed by a layer of VP2 and contains multiple copies of the RNA-dependent RNA polymerase VP1 and the mRNA-capping enzyme VP3. Double-layered particles (DLPs), representing cores surrounded by a layer of VP6, direct the synthesis of viral mRNAs. Rotavirus core- and DLP-like replication intermediates (RIs) catalyze the synthesis of dsRNA from viral template mRNAs coincidentally with the packaging of the mRNAs into the pre-capsid structures of RIs. In addition to structural proteins, the nonstructural proteins NSP2 and NSP5 are components of RIs with replicase activity. NSP2 self assembles into octameric structures that have affinity for ssRNA and NTPase and helix-destabilizing activites. Its interaction with nucleotides induces a conformational shift in the octamer to a more condensed form. Phosphate residues generated by the NTPase activity are believed to be transferred from NSP2 to NSP5, leading to the hyperphosphorylation of the latter protein. It is suspected that the transfer of the phosphate group to NSP5 allows NSP2 to return to its noncondensed state and, thus, to accept another NTP molecule. The NSP5-mediated cycling of NSP2 from condensed to noncondensed combined with its RNA binding and helix-destabilizing activities are consistent with NSP2 functioning as a molecular motor to facilitate the packaging of template mRNAs into the pre-capsid structures of RIs. Similarities with the bluetongue virus protein NS2 and the reovirus proteins sigmaNS and micro2 suggest that they may be functional homologs of rotavirus NSP2 and NSP5.

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Section: Homologs Of Nsp2 and Nsp5 For Reovirus And Bluetongue Virus mentioning

confidence: 99%

Section: Homologs Of Nsp2 and Nsp5 For Reovirus And Bluetongue Virus mentioning

confidence: 99%

Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Taraporewala

Patton

2004

Virus Research

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“…The reovirus nonstructural protein NS is a 41-kDa protein highly conserved among different reovirus strains (37,38). Initially incorrectly identified as a poly(C)-dependent RNA polymerase (39), NS is a single-stranded RNA (ssRNA)-binding protein with a reported specificity for certain regions of reovirus mRNA (40,41), although it appears to show comparable binding to viral and nonviral RNAs (42). NS has been defined as a nonclassical helicase, exhibiting strand displacement activity in the absence of ATP hydrolysis and MgCl 2 (42).…”

mentioning

confidence: 99%

Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

Zamora

Knowlton

et al. 2018

J Virol

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Viral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, using and cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the family diverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using and cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the family assort and replicate their genomes.

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“…We previously reported the purification of smal reovirus-specific particles with polycytidylate [poly(C)]-dependent RNA polymerase activity from extracts of infected cells (7). These particles were spherical or triangular with a diameter of 11 to 12 nm, and they sedimented from 13 to 19S.…”

mentioning

confidence: 99%

“…The harvest from passage 2 of the Dearing strain of reovirus (type 3, cloned [5,8]) was used as stock virus in all experiments described here. The procedures for infection and incubation of cells at 300C in the presence of actinomycin D (0.15 ,ug/ml) were as described before (5)(6)(7). For labeling of reovirus proteins, Eagle Spinner medium containing 33% of the normal complement of amino acids supplemented with 7% fetal bovine serum was used.…”

mentioning

confidence: 99%

Small Reovirus Particles Composed Solely of Sigma NS with Specificity for Binding Different Nucleic Acids

Gomatos

Prakash

Stamatos

1981

J Virol

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We reported previously that polycytidylate [poly(C)]-dependent RNA polymerase activity was a property of small spherical or triangular reovirus-specific particles which sedimented at 13 to 19S and were composed solely of the reovirus protein, sigma NS. Depending on the fraction of cellular extracts from which they were obtained, these particles exhibited marked differences in stability. Most 13 to 19S particles from a particular fraction repeatedly disaggregated into smaller 4 to 5S subunits with no enzymatic activity. Disruption of many particles could be prevented and polymerase activity retained after these particles had bound different single-stranded (ss) RNAs. Our previous results indicated that there was heterogeneity among the 13 to 19S particles in that possession of poly(C)-dependent RNA polymerase activity was a property of only some. Support for this heterogeneity was derived from the demonstration in this report that there were at least three types of binding sites present within particles in any purified preparation: (i) those binding only poly(C); (ii) those binding only reovirus ss RNAs; and (iii) those binding one or the other, but not both at the same time. It is suggested that only those particles able to bind either poly(C) or reovirus ss RNAs had poly(C)-dependent RNA polymerase activity, as reovirus ss RNAs markedly inhibited the polymerase activity. All three size classes of reovirus ss RNAs were equally effective in binding, but once bound, they were not copied. It is possible that heterogeneity in binding capacity of different particles comprised of only one protein, sigma NS, could result from the ability of subunits containing this protein to assemble into slightly different 13 to 19S particles with specificity of binding or polymerase activity conferred by the configuration of the assembled particles. The high capacity of sigma NS to bind many different nucleic acids with some specificity suggests that these particles may act during infection as condensing agents to bring together 10 reovirus ss RNA templates in preparation for double-stranded RNA synthesis.

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Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity

Cited by 21 publications

References 24 publications

Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

Small Reovirus Particles Composed Solely of Sigma NS with Specificity for Binding Different Nucleic Acids

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