Rotaviruses (RVs), members of the Reoviridae family, have genomes consisting of eleven segments of double-stranded (ds) RNA. The genome of the RV virion is contained in a non-enveloped icosahedral capsid composed of three concentric protein layers. The innermost protein layer is a smooth, thin, pseudo T=1 assembly formed from 12 decamers of the core lattice protein, VP2. Tethered to the underside of the VP2 layer are complexes comprised of the viral RNA-dependent RNA polymerase (RdRP), VP1, and the RNA-capping enzyme, VP3. Together, VP1, VP2, VP3, and the dsRNA genome form the core of the virion. The core proteins function together to transcribe the segmented dsRNA genome, producing eleven capped plus-sense (+)RNAs. The viral RdRP uses the (+)RNAs as templates for the synthesis of the dsRNA genome. Although the RdRP alone can recognize viral (+)RNAs, the polymerase is only active when VP2 is present. The VP2-dependent activity of VP1 provides a means by which genome replication (dsRNA synthesis) can be linked with genome packaging and core assembly. Newly made (+)RNAs pass from the RdRP to VP3, an enzyme which introduces m7G caps to the 5'-end of the transcripts through associated guanylyltransferase and methyltransferase activities. Genome replication and core assembly take place in cytoplasmic inclusions bodies of infected cells;these structures are referred to as viroplasms. Two viral nonstructural proteins, the octamer NSP2 and the phosphoprotein NSP5, direct the formation of viroplasms. The interactions of NSP2 and NSP5 with VP1, VP2, and VP3 coordinate genome replication and core assembly. The overriding goal of this project is to characterize the structure and function of the core proteins VP1, VP2, and VP3 and the viroplasm building-blocks NSP2 and NSP5. This includes defining the structural interfaces between the proteins and establishing how these interactions affect and regulate the activities of the proteins. Progress toward this goal in 2011-2012 is summarized below. (1) RV RNA POLYMERASES RESOLVE INTO TWO PHYLOGENETICALLY DISTINCT CLASSES THAT DIFFER IN THEIR MECHANISM OF TEMPLATE RECOGNITION. Within the Rotavirus genus, eight species (RVA-RVH) have been proposed to exist. The RVA viruses have been the most widely studied since they the primary cause of life-threatening dehydrating diarrhea in infants and young children. In collaboration with Dr. Reimar Johnes group in Germany, we determined the first known sequences for the viral RdRP (VP1)-encoding segments of RVF and RVG viruses and compared these sequences to those of other RV species. Our analysis indicates that the VP1 RNA segments and proteins resolve phylogenetically into two major clades (A and B). RVA, RVC, RVD and RVF species fall within clade A, while RVB, RVG and RVH species fall within clade B. Plus-strand RNAs of clade A viruses, and not clade B viruses, contain a 3'-proximal UGUG cassette that serves as a recognition signal mediating specific interaction with viral polymerases. The polymerase recognition signal in plus-strand RNA of clade B viruses remains to be determined. A VP1 structure for each RV species were predicted using homology modeling. Structural elements involved in interactions with the UGUG cassette were conserved among VP1 of clade A, suggesting a conserved mechanism of viral RNA recognition for these viruses. Based on the distinct characteristics of the polymerases of clade A and B RVs, including poor sequence identity values, the single RV genus should probably be resolved into two genera. Virology (2012) 431:50-57. (2) MUTATIONAL ANALYSIS OF RESIDUES INVOLVED IN NUCLEOTIDE AND DIVALENT CATION STABILIZATION IN THE RV RdRP CATALYTIC POCKET. The RV RdRP, VP1, contains canonical RdRP motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential importance to nucleotide and divalent cation stabilization, we aligned VP1 of divergent RVs and the structural homolog reovirus lambda3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRP motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRP activity to particle assembly. Virology (2012) 431:12-20 (3) PREDICTED STRUCTURE AND DOMAIN PURIFICATION OF RV CAPPING ENZYME VP3. VP3 is a critical RV enzyme that is present in virions at low copy number. Several activities involved in viral RNA capping, including guanylyltransferase (GTase) and N7 and 2'-O methyltransferase (MTase), have been ascribed to VP3. The structure of the 835-amino acid VP3 protein, however, remains unknown. Based on homology modeling with the bluetongue virus (BTV)-capping enzyme, VP4, the structure of RV VP3 residues 109-634 was predicted. RV VP3 and BTV VP4 appear to share a similar modular organization, with the 2'-O MTase domain positioned within the N7 MTase domain. In addition, RV VP3 and BTV VP4 have in common several putative MTase active-site residues. The structure of VP3 residues 697-800 was modeled based on homology with members of the 2H phosphoesterase superfamily of enzymes and appears to contain two conserved active-site His-X-Thr/Ser motifs. No homology models were generated with confidence for the N-terminal 108 residues of VP3. Based on predicted structure, we have expressed individual domains of VP3 in E. coli as N-terminal fusions with maltose binding protein. We utilized amylose affinity chromatography to purify fusion constructs representing the predicted N-terminal, 2'-O MTase, combined N7 and 2'-O MTase, and GTase and 2H phosphoesterase domains. The affinity tag was removed by cleavage at an engineered tobacco etch virus protease site. Currently, we are characterizing the solubility and activities of the purified VP3 fragments and working to determine their structures. (4) PROBING NSP5 FUNCTION VIA CHARACTERIZATION OF THE RV TEMPERATURE-SENSITIVE (ts) MUTANT, tsJ. The RV 28kD phosphoprotein NSP5 is an essential building block of viroplasms. NSP5 interacts with NSP2 and VP2 and binds non-specifically to viral RNA. However, the importance of these interactions and the significance of NSP5 phosphorylation in the viral replication cycle are poorly understood. The RV mutant, tsJ, contains a ts lesion that maps to the NSP5-encoding segment. At the non-permissive temperature, the ts lesion causes a 10ex3 reduction in virus titer. The ts lesion is correlated with an alanine to glycine substitution at residue 182 of NSP5. In this study, we analyzed the tsJ phenotype to better understand the function of NSP5. Our results indicate that the level of NSP5 expression in tsJ-infected cells was two-fold higher at non-permissive temperature than at permissive temperature. No similar increase was seen in the expression of NSP2 and VP6. At the non-permissive temperature, viroplasms were initially formed in tsJ-infected cells, but by 9 hours post-infection few inclusions were detected. Additionally, the disappearance of viroplasms was concurrent with mislocalization of NSP2, VP2, and tsJ NSP5. By taking advantage of the reduced replication phenotype of tsJ at the non-permissive temperature, we are currently developing an NSP5-dependent complementation assay. Together, characterization of tsJ and the development of a NSP5 complementation assay will allow for elucidation of NSP5 functions during RV infection.
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