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 inclusion 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 2012-2013 is summarized below. (1) IDENTIFICATION OF RESIDUES IMPORTANT FOR THE FUNCTION OF THE NTP ENTRY TUNNEL OF THE ROTAVIRUS RNA POLYMERASE. The active site of the RV RdRP, VP1, is accessible via four tunnels. One of these, the nucleotide entry tunnel, is proposed to regulate the flow of nucleotides and divalent cations in and out of the active site during catalysis. The X-ray crystallography structure of VP1 indicates that its nucleotide entry tunnel is flanked by an inherently flexible loop. To evaluate the significance of the loop to the exchange of nucleotides and divalent cations, we compared the structure of VP1 to those of several other viral RdRPs. The analysis suggests that the presence and flexibility of the loop is conserved among viral RdRPs, along with a number of positively charged (e.g., lysine, arginine) residues. To better define the importance of the loop, a collection of mutant VP1 proteins were generated that contained deletions or substitutions in the loop;the polymerase activity of the mutant proteins were then determined by in vitro assay. The results showed that the length and overall structure of the loop and the presence of conserved positively charged residues were all important for polymerase stability and/or efficient RNA synthesis. Thus, the loop and its residues may have important roles in regulating substrate exchange and efficient nucleotide incorporation. (2) LOCATION OF DOMAINS AND RESIDUES IN THE ROTAVIRUS VP3 PROTEIN THAT FUNCTION IN RNA CAPPING. Several activities involved in viral RNA capping, including guanylyltransferase (GTase) and N7 and 2'-O methyltransferase (MTase), have been ascribed to the 835-amino acid VP3 protein. Based on homology modeling with the bluetongue virus capping enzyme, we predicted the structure of RV VP3 residues 109-634. The N-terminal domain of this region, whose function is unknown, is followed by the 2'-O MTase domain inserted within the N7 MTase domain;this is the followed by the GTase domain. Based on sequence alignment of RVs from divergent species, we have identified highly conserved motifs. These motifs align with putative active sites and ligand-binding pockets in the predicted MTase domains and in a specific region of the predicted GTase domain. Sequence-based mutagenesis of the predicted GTase domain in combination with biochemical analyses of recombinant VP3 mutants has permitted identification of residues that are involved in VP3 autoguanylation. These findings provide additional support for our VP3 structural model. (3) DISCOVERY OF AN ANTAGONIST OF THE OAS/RNASE L PATHWAY AT THE C-TERMINUS OF THE ROTAVIRUS CAPPING ENZYME VP3. The structure of the C-terminal domain (residues 697-835) of the 835-amino acid VP3 was modeled based on homology with members of the 2H phosphoesterase superfamily of enzymes and appeared to contain two conserved active-site His-X-Thr/Ser motifs. The coronavirus accessory protein ns2, which is also predicted to be a member of the 2H phosphoesterase superfamily, antagonizes the oligoadenylate synthetase (OAS)-ribonuclease (RNase) L pathway, an important arm of the host innate immune response, by cleaving 2,5-oligoadenylates (2-5A). In collaboration with Susan Weiss (University of Pennsylvania) and Robert Silverman (Cleveland Clinic), we found that the VP3 C-terminal domain can similarly cleave 2-5A in vitro. We engineered the coronavirus mouse hepatitis virus (MHV) to express the VP3 C-terminal domain and found that this region of VP3 could complement the attenuation caused by mutation of the ns2 active site both in tissue culture and a mouse model of infection. Thus, in addition to its capping functions, VP3 appears to antagonize the host innate immune response to viral infection by degrading the 2-5A moiety that activates the RNase L pathway. This activity of VP3 likely explains why earlier studies indicated that VP3 could function as a virulence factor. (R Zhang, BK Jha, KM Ogden, B Dong, L Zhao, R Elliott, JT Patton, RH Silverman, SR Weiss (2013) Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity. Proc Natl Acad Sci USA 110:13114-13119).

Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2013
Total Cost
$571,322
Indirect Cost
City
State
Country
Zip Code
Ogden, Kristen M; Snyder, Matthew J; Dennis, Allison F et al. (2014) Predicted Structure and Domain Organization of Rotavirus Capping Enzyme and Innate Immune Antagonist VP3. J Virol 88:9072-85
Zhang, Rong; Jha, Babal K; Ogden, Kristen M et al. (2013) Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity. Proc Natl Acad Sci U S A 110:13114-9
Navarro, Aitor; Trask, Shane D; Patton, John T (2013) Generation of genetically stable recombinant rotaviruses containing novel genome rearrangements and heterologous sequences by reverse genetics. J Virol 87:6211-20
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