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

Abstract

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.

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

“…The NS protein, originally called essential noncapsid protein 2A (57), was first identified as a nucleic acid-binding protein in cellular extracts derived from reovirusinfected cells (40). NS binds nucleic acids with positive cooperativity and covers a length of ϳ25 to 27 nucleotides at saturation (42).…”

“…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).…”

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

“…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).…”

Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3

“…Binding of recombinant bluetongue virus NS2 to RNA does not seem to be sequence specific (48,54). Sigma NS purified from mammalian reovirus-infected cells reportedly prefers reovirus 3Ј-end mRNA (16,42), while avian reovirus-expressed (51) and E. coli-expressed sigma NS (39) do not show any specificity. Limited mutations outside the conserved motif, as well as deletion of the entire motif (47), abolished RNA binding by NS2 (15,54).…”