Scholarship & Research
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/1
Browse
5 results
Search Results
Item The effects of the bovine respiratory syncytial virus on the ciliated epithelium of fetal bovine tracheal organ culture(Montana State University - Bozeman, College of Agriculture, 1979) Cantrell, Charles GarrettItem The characterization of two bovine adenoviruses isolated from calves exhibiting clinical and pathological signs of weak calf syndrome(Montana State University - Bozeman, College of Agriculture, 1979) Shadoan, Patricia KnoxItem The role of NSP1 in the regulation of rotavirus gene expression(Montana State University - Bozeman, College of Agriculture, 2003) Mitzel, Dana Nicole; Chairperson, Graduate Committee: Michele Hardy.Item Rotavirus NSP1 is an interferon system antagonist(Montana State University - Bozeman, College of Agriculture, 2008) Graff, Joel Wallace; Chairperson, Graduate Committee: Michele Hardy; Mark Jutila (co-chair)Rotaviruses cause severe gastroenteritis in mammals, including humans and livestock. Most rotavirus proteins have known functions, either as a mediator of virus replication or as a component of the infectious virus particle. The function of nonstructural protein 1 (NSP1) was unknown. However, it has been suggested that the function of NSP1 involved interactions with cellular proteins. Using the NSP1 encoded by a bovine rotavirus as the bait protein of a yeast-two hybrid interaction trap, interferon regulatory factor 3 (IRF3) was identified as an NSP1-interacting protein. Due to the importance of IRF3 in initiating an interferon response, we hypothesized that NSP1 acts to antagonize the interferon system. A comprehensive set of experiments yielded the following observations. Interferon-beta (IFNbeta) induction was blocked in wild-type, but not NSP1 null, infected cells. Expression of NSP1 in the absence of infection resulted in proteasome-mediated degradation of IRF3. A cysteine-rich zinc-binding region near the amino-terminus of all known NSP1 sequences resembles a domain found in hundreds of E3 ubiquitin ligases. Mutational analysis of the zinc-binding domain was consistent with NSP1 acting as an E3 enzyme. The NSP1 of a murine strain of rotavirus also interacted with IRF3. The NSP1 proteins encoded by the bovine and murine rotavirus strains showed low sequence homology (37% identity), which indicated that directing degradation of IRF3 may be a common property of NSP1 proteins from many different rotavirus strains. Screening of simian, bovine, and porcine rotavirus strains indicated that the porcine strain OSU was the only wild-type strain that was unable to direct IRF3 degradation. Characterization of OSU showed that this strain was able to block IFNbeta induction by antagonizing NFKB signaling, rather than IRF3 signaling. While analyzing NFKB signaling in rotavirus infected cells, we found that the F box protein, betaTrCP, of the Skp1/Cul1/F box multi-subunit E3 enzyme was targeted for degradation by OSU NSP1 and the NSP1 of a bovine rotavirus strain. Together, the results presented in this dissertation have determined that rotavirus NSP1 functions as an interferon antagonist by directing the proteasome-mediated degradation of IFNbeta induction signaling components, most likely by acting as an E3 ubiquitin ligase.Item The role of VPg in translation of calicivirus RNA(Montana State University - Bozeman, College of Agriculture, 2005) Daughenbaugh, Katie Finney; Chairperson, Graduate Committee: Michele Hardy.Molecular mechanisms of Norovirus replication remain for the most part undefined, primarily due to the lack of cell culture and small animal model systems. However, sequence comparisons and studies using cultivable caliciviruses have lead to the description of many features of the viral genome. Genomes are positive sense RNA, where the genome itself serves as mRNA for the production of viral protein. Additionally, viral RNA is covalently attached at the 5α end to the viral protein VPg. VPg is required for infectivity of the RNA by transfection, and removal of VPg by proteinase K treatment reduces the ability of the RNA to be translated in vitro. Because of these data, and because viral RNA is presumably not translated by an IRES mechanism, it has been suggested that VPg plays a role in translation of viral RNA. Studies described herein were initiated to investigate the potential role for Norwalk virus (NV) VPg in this process. It was found that NV VPg binds translation initiation factor 3 (eIF3) directly and in cell lysates, and is present in complexes with other eIFs including the cap-binding protein eIF4E, the large scaffolding protein eIF4G, the S6 ribosomal protein, and eIF2á, a component of the ternary complex. VPg also inhibits translation of reporter RNAs in vitro, suggesting that the interactions observed between VPg and eIFs are relevant to translation. Regions of VPg responsible for interactions with eIFs were mapped, and it was found that interaction between VPg and the 40S ribosome is most likely that which is responsible for translation inhibition of the reporter RNAs. VPg directly binds 40S ribosomal subunits by sucrose density gradient centrifugation, and this interaction is likely mediated by the central domain of VPg, similar to binding properties observed for the universally conserved factor eIF1A. Finally, a recently discovered, cultivable murine norovirus ₁ 1 (MNV-1) was used to ask if interactions between VPg and eIFs occur in infected cells. It was found that VPg of MNV ₁ 1 coprecipitates with eIF4GI, the d subunit of eIF3, and eIF4E from infected cells, and that this VPg has similar binding properties as the NV VPg. Together the data support the hypothesis that VPg plays a role in translation of viral RNA during infection, and suggets a third mechanism of ribosome recruitment dependent upon protein-protein interactions between VPg and eIFs. These studies also highlight the possibility of using MNV ₁ 1 as a molecular model for the study of human norovirus infection.