The mammalian orthoreovirus bicistronic M3 mRNA initiates translation using a 5' end-dependent, scanning mechanism that does not require interaction of 5'-3' untranslated regions.

Mammalian orthoreovirus mRNAs possess short 5' UTR, lack 3' poly(A) tails, and may lack 5' cap structures at late times post-infection. As such, the mechanisms by which these viral mRNAs recruit ribosomes remain completely unknown. Toward addressing this question, we used bicistronic MRV M3 mRNA to analyze the role of 5' and 3' UTRs during MRV protein synthesis. The 5' UTR was found to be dispensable for translation initiation; however, reducing its length promoted increased downstream initiation. Modifying start site Kozak context altered the ratio of upstream to downstream initiation, whereas mutations in the 3' UTR did not. Moreover, an M3 mRNA lacking a 3' UTR was able to rescue MRV infection to WT levels in an siRNA trans-complementation assay. Together, these data allow us to propose a model in which the MRV M3 mRNA initiates translation using a 5' end-dependent, scanning mechanism that does not require the viral mRNA 3' UTR or 5'-3' UTRs interaction.

Formation of the factory matrix is an important, though not a sufficient function of nonstructural protein mu NS during reovirus infection.

Genome replication of mammalian orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein microNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, microNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for microNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (lambda1, lambda2, lambda3, micro2, and sigma2) and nonstructural protein sigmaNS, can associate with microNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in microNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log(10) values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement microNS expression and to rescue infectious yields by ~100-fold. We next used microNS mutants specifically defective at forming factory-matrix structures to show that this function of microNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of microNS prevented rescue of viral growth while causing microNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of microNS, designed to represent microNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of microNS known to be involved in both micro2 and sigmaNS association. Thus, factory-matrix formation is an important, though not a sufficient function of microNS during MRV infection; microNS is multifunctional in the course of viral growth.

Guanidine hydrochloride inhibits mammalian orthoreovirus growth by reversibly blocking the synthesis of double-stranded RNA.

Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2C(ATPase), resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on mammalian orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family Reoviridae. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.

Replication of poliovirus RNA with complete internal ribosome entry site deletions.

cis-acting RNA sequences and structures in the 5' and 3' nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5' nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative- and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES.

Nucleoside and RNA triphosphatase activities of orthoreovirus transcriptase cofactor mu2.

The mammalian Orthoreovirus (mORV) core particle is an icosahedral multienzyme complex for viral mRNA synthesis and provides a delimited system for mechanistic studies of that process. Previous genetic results have identified the mORV mu2 protein as a determinant of viral strain differences in the transcriptase and nucleoside triphosphatase activities of cores. New results in this report provided biochemical and genetic evidence that purified mu2 is itself a divalent cation-dependent nucleoside triphosphatase that can remove the 5' gamma-phosphate from RNA as well. Alanine substitutions in a putative nucleotide binding region of mu2 abrogated both functions but did not affect the purification profile of the protein or its known associations with microtubules and mORV microNS protein in vivo. In vitro microtubule binding by purified mu2 was also demonstrated and not affected by the mutations. Purified mu2 was further demonstrated to interact in vitro with the mORV RNA-dependent RNA polymerase, lambda3, and the presence of lambda3 mildly stimulated the triphosphatase activities of mu2. These findings confirm that mu2 is an enzymatic component of the mORV core and may contribute several possible functions to viral mRNA synthesis.

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis.

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.