Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: The initial response of cells to virus infection is to activate a complex signaling cascade that results in induction of a specific pattern of gene expression to establish an antiviral state that restricts virus replication and spread. Viruses have evolved to interfere with multiple steps required to activate this cell intrinsic, or innate, immune response. The overall goals of this project were to define cellular proteins involved in the antiviral interferon (IFN) signaling pathway that are directly affected by rotavirus replication, and to determine how infection inhibits their function. We sought to test the hypothesis that rotaviruses target multiple steps in induction and amplification of IFN pathways. We and others have identified interferon regulatory factors (IRFs) as targets of rotavirus nonstructural protein NSP1, which is now established as an IFN antagonist and predicted to be a viral E3 ubiquitin ligase. We focused the current studies on porcine rotavirus OSU and NSP1 encoded by this strain. The objectives were to define the mechanism of IFN resistance for OSU. We performed a series of experiments aimed toward NSP1 specifically, and defined cellular proteins modulated by OSU in the context of the IFN response. The activities and products linked to this project include basic research on fundamental mechanisms of viral interactions with the host defense response relevant to an important agent of neonatal diarrhea in domestic livestock. The results of these basic studies are important to animal health because in order to enhance an effective first defense response to viral infection, molecular details of the pathways that are regulated must be understood. We performed experiments using techniques of cell and molecular biology, and state-of-the art proteomics to assess interactions between NSP1 and cell proteins key to efficient induction of IFN. Results were disseminated through presentation at the annual American Society for Virology meeting and through publications in 2009 and 2011. Significantly, our findings subsequently have been confirmed by others, and recognized by citation in several recent reviews of viral IFN antagonism. PARTICIPANTS: Dr. Michele Hardy Project Director: Dr. Hardy oversees the entire project, assists with experimental design and interpretation, manuscript and progress report preparation, and presentation of data. Dr. Khalil Ettayebi, Assistant Research Professor: Dr Ettayebi performed proteomics experiments specifically related to the goals of the project. Nicholas Faunce, Research Associate: Mr Faunce performed experiments directly related to the project including analysis of the UPR and assistance with proteomic data analysis and experimental confirmation. Dr. Jose Luis Zambrano, Postdoctoral Associate: Dr. Zambrano performed experiments to characterize the UPR in rotavirus infected cells. Jesse Mostipak, Graduate Student: Ms. Mostipak performed experiments related to the IFN response in rotavirus infected cells. Dr. Joel Graff, Graduate Student (former): Dr. Graff initiated and reported the studies on IFN antagonism by NSP1. John Anthony Junghans, Undergraduate Student: Performed studies to establish NSP1 purification methods. Jay M Hendricks, Research Associate: Laboratory Manager; assists on all ongoing research projects and participates in training of all laboratory personnel through technical and scientific supervision. TARGET AUDIENCES: Target audience is researchers at all levels in virology, specifically including those studying innate immune responses to virus infection. PROJECT MODIFICATIONS: The scientific goals of the project did not change. However, we utilized a primary approach of DIGE for proteomics studies that was presented and proposed as a parallel technique in the original application.
Impacts
We discovered that NSP1 inhibits the activity of cellular protein beta-transducin repeat protein (Beta-TrCp) that is required for activation of transcription factor NFkappaB. NFkappaB has multiple functions in the cell and controls cell proliferation, differentiation, survival and inflammatory responses. The ability of NSP1 to down-regulate NFkappaB by this mechanism is unique not only to rotavirus but also is the first report of this mechanism of IFN antagonism. Our extensive global analysis of differential gene expression in OSU infected, IFN treated, and OSU-IFN treated cells, resulted in the discovery that the unfolded protein response (UPR), a prominent cellular pathway that functions in maintenance of cellular homeostasis, was activated upon initial infection with OSU, but then down-regulated early post-infection. The UPR is a stress-related response activated upon accumulation of misfolded protein in the ER, and functions in part by slowing global cellular protein synthesis. If the stressor cannot be resolved, the cell is induced to undergo apoptosis. Thus it is advantageous to the virus to down-regulate the consequences of UPR activation so that normal cell functions including protein synthesis are maintained. Our data were published as the first report of UPR modulation by rotavirus, and a subsequent publication by others confirmed our findings. In the final stages of the project, we again sought to identify cellular proteins targeted by NSP1 for modulation. Our preliminary data suggest interactions between NSP1 and a novel signaling protein only recently reported to play a role in the induction of the antiviral IFN response, and to date has not been reported as being modulated by virus infection. We continue our investigations into the function of this protein and what effects NSP1 expression might have on its normal function.
Publications
- Graff, J.W., K Ettayebi and M.E. Hardy 2009. Rotavirus NSP1 inhibits NFkappaB activation by targeting Beta-TrCP for degradation: a novel mechanism of IFN antagonism. PLoS Pathogens 5(1): eoi:10.1371/journal.ppat.1000280.
- Sen, A., N. Feng, K. Ettayebi, M.E. Hardy, and H.B. Greenberg. 2009. IRF3 inhibition by rotavirus NSP1 is host cell and viral strain independent, but independent of NSP1 proteasomal degradation. J Virol. 83:10322-10335.
- Zambrano, J.L, K. Ettayebi, W. Maaty, Faunce, N.R., Bothner, B. and M.E. Hardy. 2011. Rotavirus infection activates the UPR but modulates its activity. Virology J. 8:859
|
Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: During this reporting period, we completed our studies on comparative changes in a subset of the cellular proteome in response to porcine rotavirus OSU infection and treatment with IFN. We performed biochemical and microscopy experiments and analyses that generated data to support our preliminary conclusions from the proteomic analysis, suggesting rotavirus infection modulates the ER-mediated unfolded protein response (UPR). Data from these efforts allowed us to expand the work to specifically understand how porcine rotavirus modulates cellular stress responses, including the UPR and IFN systems. The data were presented at the USDA/NIFA annual Project Director's meeting and have been published. PARTICIPANTS: Dr Michele Hardy - PD, experimental design, development of proposal, hypothesis, manuscript preparation, supervision; Dr Khalil Ettayebi - Assistant Research Professor, experimental design and execution, data analysis, manuscript preparation; Dr Jose Luis Zambrano - experimental design and execution, data analysis, manuscript preparation; Nicholas R Faunce - Research Associate - experimental design and execution; Jay Hendricks- Research Associate - experimental design and execution. TARGET AUDIENCES: Researchers, students and teachers in virology, diarrheal diseases, immunology, infectious diseases, viral immune evasion strategies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Mechanisms utilized by viruses to evade cellular antiviral responses are receiving increased attention because of the potential to develop attenuated vaccines consisting of viruses with weakened evasion strategies, or to target viral protein antagonists for antiviral drug development. Viruses from several families important to U.S. agriculture and public health including bovine viral diarrhea virus, West Nile virus, influenza virus, rotavirus, SARS, and classical swine fever virus, among many others, all encode proteins that function to down-regulate the host antiviral response. Rotavirus infections cause life-threatening acute diarrhea in newborns of domestic livestock, including bovine and swine species. We have shown that nonstructural protein NSP1 functions at least in part as an innate immune antagonist. During this reporting period, we performed experiments directly related to the funded objectives. We completed an extensive proteomics study on OSU infected cells and IFN treated cells to determine what cellular proteins and responses are modulated by either virus, IFN or both, and whether any of these responses could be attributed to NSP1. The data suggested OSU significantly modulates the unfolded protein response (UPR) in the ER of cells that responds to stress and maintains cellular homeostasis under steady-state conditions. Our studies suggest a prominent modulation of the UPR, initially observed by changes in the levels of multiple heat shock proteins. Follow-up experiments using protein and confocal microscopy analyses confirmed that although the UPR was induced during rotavirus infection, its functional effects were attenuated. While the molecular mechanisms by which this occurs still are not clear, they are under investigation and do not seem to correlate with NSP1 expression. Instead, the data point toward a sequestration of UPR proteins in concentrated areas of virus replication in the cell, by mechanisms that may involve direct interactions with viral proteins. These data are important because thus far, a broad picture of the effects of infection on cell stress pathways remains largely undefined. Our published data recently were confirmed by another group.
Publications
- Zambrano, J.L., K Ettayebi, W.S. Maaty, N.Faunce, B. Bothner, and M.E. Hardy. 2011. Rotavirus infection activates the UPR but modulates its activity. Virol J., Jul 20:8:359
|
Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: During this reporting period (09/01/08-08/31/09), we continued experiments directly related to the funded objectives. We completed the experimental portion of the proteomics study and are now analyzing the data to determine what proteins are differentially regulated by either porcine rotavirus infection or by IFN. Previous data suggested the cellular Unfolded Protein Response (UPR) was down-regulated in OSU infected cells, and follow up experiments were performed to confirm these data. Data from the proteomics efforts have allowed us to expand the work to specifically understand how porcine rotavirus modulated cellular stress responses, including the IFN system. Proteomics data were presented to the international virology community at the 29th Annual Meeting of the American Society for Virology in Bozeman, MT. PARTICIPANTS: Dr Michele Hardy Project Director Dr Hardy oversees the entire project, assists with experimental design and interpretation, manuscript and progress report preparation, and presentation of data. Dr Khalil Ettayebi Assistant Research Professor Dr Ettayebi performed proteomics experiments specifically related to the goals of the project. Nicolas Faunce Research Associate Mr Faunce performed experiments directly related to the project including analysis of the UPR. Dr Jose Luis Zambrano Postdoctoral Associate Dr Zambrano performed experiments to characterize the UPR in rotavirus infected cells Jesse Mostipak Graduate Student Ms Mostipak performed experiments related to the IFN response in rotavirus infected cells TARGET AUDIENCES: Target audience is researchers at all levels in virology, specifically including those studying innate immune responses to virus infection. PROJECT MODIFICATIONS: No major changes
Impacts
The Unfolded Protein Response (UPR) is a normal cellular response to stress that is evoked by the presence of unfolded or misfolded protein accumulation in the ER. The UPR is commonly induced in response to virus infection, thus our proteomics data that suggested modulation of cellular proteins important in the UP was not surprising. It was unexpected however, that most of the UPR proteins identified as differentially regulated by porcine rotavirus infection were shown to be decreased. This is in contrast to most published data demonstrating similar proteins were up-regulated. These data are new to the field and have potential to change current concepts regarding how the UPR can be regulated in rotavirus infected cells.
Publications
- K Ettayebi, M. Shaneyfelt, J.L. Zambrano, W. Maaty, B. Bothner, and M.E. Hardy. 2010. A proteomics approach to identify cellular proteins modulated by rotavirus infection. 29th Annual Meeting of the American Society for Virology. July 17-21st, 2010, Bozeman, MT.
|
Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Mechanisms utilized by viruses to evade cellular antiviral responses are receiving increased attention because of the potential to develop attenuated vaccines consisting of viruses with weakened evasion strategies, or to target viral protein antagonists for antiviral drug development. In addition, proposed strategies to amplify the innate response to enhance natural disease resistance to infection are increasing in number. Generally, induction of type I interferons (IFN) and consequent expression of a pattern of IFN-responsive genes are the targets of viral innate immune antagonists. Viruses from several families important to U.S. agriculture and public health including bovine viral diarrhea virus, West Nile virus, influenza virus, rotavirus, SARS, and classical swine fever virus, among many others, all encode proteins that function to down-regulate the host antiviral response. Rotavirus infections cause life-threatening acute diarrhea in newborns of domestic livestock, including bovine and swine species. We and others have shown that nonstructural protein NSP1 functions at least in part as an innate immune antagonist. We have predicted that nonstructural protein NSP1 from different rotavirus strains targets a range of cellular substrates that play essential roles in initiation of antiviral signaling pathways. We recently have demonstrated at least two mechanisms by which rotavirus strains inhibit the initial lines of host defense. The objectives of this proposal are to define the mechanism of IFN resistance for porcine rotavirus OSU. We proposed to define how OSU and OSU NSP1 expression attenuate the IFN response by investigating the effects on NFkB activation and adaptor protein RACK1 degradation. Second, we proposed to investigate mechanisms of NSP1-mediated resistance to IFN by defining target substrates using state-of-the-art proteomics approaches. During this reporting period (09/01/08-08/31/09), we performed experiments directly related to the funded objectives. We performed extensive proteomics study on OSU infected cells as well as IFN treated cells to determine what cellular proteins and responses are modulated by either virus, IFN or both. The data suggest OSU significantly modulates the unfolded protein in the ER of cells that responds to stress as well as maintaining cellular homeostasis under steady-state conditions. Although we have not yet defined a role for RACK1, studies suggest multiple signaling pathways are affected by OSU infection, and the ubiquitous nature of RACK1 in regulating protein kinases strongly supports a functional role of this protein. We have expanded our investigations to include alternate targets and signaling pathways to complement those originally proposed. PARTICIPANTS: Dr Michele Hardy - PD, experimental design, development of proposal, hypothesis, manuscript preparation, supervision; Dr Khalil Ettayebi - Assistant Research Professor, experimental design and execution, data analysis, manuscript preparation; Joel Graff - Graduate Student, experimental design and execution, data analysis, manuscript preparation; Jose Luis Zambrano - experimental design and execution, data analysis; Nick Faunce - Research Associate - experimental design and execution; Jay Hendricks- Research Associate - experimental design and execution TARGET AUDIENCES: Researchers in virology, diarrheal diseases, immunology, infectious diseases, viral immune evasion strategies PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our studies suggest a prominent modulation of the cellular unfolded protein response during OSU infection. These data were derived from our proteomics studies, where multiple heat shock proteins were found to be up- or down-regulated in response to infection. These data are important because thus far, a broad picture of the effects of infection on cell stress pathways remains largely undefined.
Publications
- Graff, J.W., K Ettayebi and M.E. Hardy 2009. Rotavirus NSP1 inhibits NFκB activation by targeting β-TrCP for degradation: a novel mechanism of IFN antagonism. PLoS Pathogens 5(1): eoi:10.1371/journal.ppat.1000280.
|
|