The objectives of this project, and related progress in the past year, are reviewed below. (1) DEVELOPMENT OF A REVERSE GENETICS SYSTEM THAT CAN BE USED TO MODIFY THE ANTIGENICITY AND VIRULENCE OF ROTAVIRUSES (RVS). While an efficient single-gene reverse genetics system has been described for the RVs, a fully recombinant reverse genetics system that allows engineering of any of the eleven RV genes has yet to be developed. Towards establishing such a system, we prepared capped full-length T7 transcripts of each of the eleven viral genes by in vitro transcription. The transcripts were co-transfected into cells following reverse genetics protocols established for bluetongue virus, which like RV is a member of the Reoviridae family. However, these experiments have so far failed to generate recombinant RVs. Similarly, co-transfection of T7-driven expression vectors encoding full-length RV genes into cells infected with vaccinia viruses that produce T7 RNA polymerase failed to generate recombinant RVs. (2) ELUCIDATION OF RV MECHANISMS OF SUBVERTING HOST ANTIVIRAL PATHWAYS. (a) DEGRADATION OF BETA-TRCP BY NSP1. The RV nonstructural protein NSP1 inhibits the expression of type I interferon (IFN), thereby promoting viral replication, spread, and pathogenesis. NSP1 is predicted to function as an E3 ubiquitin ligase, inducing polyubiquitination and proteasome-mediated degradation of the IFN-regulatory factors IRF3, IRF5, IRF7 and IRF9. Select RV strains inhibit IFN by an alternate route, in which NSP1 mediates the degradation of b-TrCP, a member of the SCF family of E3 ubiquitin ligases. b-TrCP regulates a number of cellular transcription factors by recognizing a conserved phosphorylated epitope on its targets, among which is the NF-kappa-B (NFkB) inhibitor I-kappa-B-alpha (IkBa). NFkB resides in the cytoplasm in an inactive complex with IkBa, until pathogen detection triggers phosphorylation of IkBa and its degradation by b-TrCP. NFkB then translocates to the nucleus, where it upregulates expression of IFN and other genes involved in the host immune response. Among NSP1 proteins that degrade or otherwise fail to upregulate b-TrCP upon co-expression, we identified a conserved epitope in the C-terminus that mimics the b-TrCP recognition site on IkBa. Further phylogenetic analysis revealed conservation of this epitope in the NSP1 protein of most human RV strains, whereas most animal RV strains lack it. Mutating the epitope to disrupt predicted phosphorylation sites blocked degradation of b-TrCP by NSP1, whereas NSP1 mutants that mimicked phosphorylation retained degradation activity. b-TrCP interacts with target proteins through its C-terminal WD40 domain and we found that this region was necessary and sufficient for degradation by NSP1. A b-TrCP mutant unable to bind and degrade phosphorylated IkBa also could not be degraded by NSP1. Transferring the epitope to an NSP1 protein that could not previously degrade b-TrCP imparted degradation activity. These results indicate that NSP1 can mimic the interaction of b-TrCP with cellular proteins to bind and degrade this key factor in NFkB activation. This strategy may have evolved in human RV strains and is distinct from the targeted degradation of IRFs by most animal RVs. (b) STRATEGY FOR EXPRESSION OF RV RECOMBINANT NSP1. Attempts to biochemically and structurally characterize NSP1 have been hampered by an inability to express the recombinant protein efficiently and in soluble form. Limited characterization has demonstrated NSP1 binding to zinc (presumably through a predicted RING finger) and RNA through a relatively conserved N-terminus, and IRF proteins through a poorly conserved C-terminus. We determined a method for high-yield production of soluble rotavirus NSP1 fragments in Escherichia coli (E. coli). By fusing the E. coli maltose-binding protein (MBP) to the N-terminus of NSP1 (MBP-NSP1), we produced NSP1 fragments that encompass the predicted RING finger and RNA binding regions. The efficiency of MBP-NSP1 expression was independent of induction temperature and IPTG concentration, and did not require supplementation of cultures with zinc. Fusing a second, hexahistidine tag to the MBP N-terminus enabled us to purify these fragments to near homogeneity. These fragments were stable in solution and were monomeric by size exclusion chromatography. Although expression of full-length NSP1 remains a challenge and will necessitate further optimization of this strategy, we can begin to rigorously address the molecular interactions currently attributed to (zinc, RNA) and predicted for (E2 ubiquitin conjugating enzyme) NSP1, thereby revealing the full extent of its role during infection. (3) ANALYSIS OF THE DIVERSITY AND EVOLUTION OF THE RV GENOME. LARGE SCALE ANALYSIS OF THE PATTERNS INFLUENCING RV REASSORTMENT. Two major genome constellations define RVs responsible for disease in humans, suggesting these gene combinations may be specifically optimized for infection and replication in this animal species. Pure Wa-like strains, characterized by an I1-R1-C1-M1-A1-N1-T1-E1-H1 genome constellation, are the most common cause of RV disease in humans, and compose nearly 80% of the genomes fully sequenced to date. A distant second, the pure DS-1 like strains, characterized by an I2-R2-C2-M2-A2-N2-T2-E2-H2 genome constellation, represent only 9%. Reassortants, which are sporadically isolated from humans, provide insight into the limitations and allowances shaping these two constellations. Strains infecting animals appear to tolerate a different range of constellation diversity and occasionally donate novel segments to human strains. However, the factors influencing the potential for these events in the context of natural infection remains elusive. Phylogenetics provides an analytical approach for identifying and assessing the frequency of reassortment between commonly circulating strains and novel gene segments. To gain insight into possible patterns of reassortment, we curated a collection of 480 human and animal strains, for which all eleven genes had been sequenced and made available through Genbank. Pairwise distance methods were used to calculate the intragenotypic amino acid diversity between established constellations and the range of diversity for RVs infecting a single host. Bayesian Tip Significance Testing, BaTS, can be used to statistically correlate a character of interest, be it species or constellation, with phylogeny. A null hypothesis, assuming no correlation between traits and phylogeny, is used to generate a predicted distribution. By comparing this value to the observed distribution, the frequency of reassortment between genetic backbones can be compared. Similarly, host origin can be mapped to identify which genes are more likely host determinants. Compared with strains isolated from other species, the serotypes of human RVs exhibit a high level of VP7 diversity and a very low level of VP4 diversity, which may reflect the balance between immunological pressure on VP7 and the need to conserve VP4 function in entry into human cells. Certain genotype 2 segments, isolated from reassortant strains, appear to be phylogenetically distinct from those expressed in pure DS-1-like constellations. Further analysis of these genotypic changes in structural context suggests that a specific combination of residues may constitute a phenotype uniquely suited for incorporation into atypical backbones. Of the genes analyzed thus far by BaTS, NSP1 exhibits a particularly strong correlation with host species. The determinants governing viable reassortment among rotavirus define the basic biology and evolution of these pathogens, yet they remain to be identified. Extending the understanding of these patterns could inform the design of stable vaccines capable of attenuating the course of infection.
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