Research into mechanisms utilized by viruses to evade cellular antiviral responses is receiving increased attention. These pathways and the key players, both viral and host, have garnered such focus because of the potential to develop attenuated vaccines consisting of viruses with weakened evasion strategies. Inducers of type I interferons (IFN) typically are targets of innate immune antagonists. The complexity of the pathways suggests there is much to learn about the regulation of innate immune signals and the viral mechanisms that have evolved to escape these responses. Rotaviruses are the major cause of acute viral gastroenteritis in children under 5 years of age. Two attenuated vaccines recently have been licensed for use in some countries, yet it remains unknown how poor nutritional status or concurrent parasitic infections will affect responses to the vaccines. Thus it is important to understand how the innate antiviral response is triggered in rotavirus infected cells and what viral proteins function to modulate this first line of host defense. An understanding of virus-host interactions at the early stages of infection will establish the basics for new approaches to enhance the innate response in ways that bypass viral evasion strategies. We identified an interaction between nonstructural protein NSP1 and IFN-regulatory factor 3 (IRF3), a transcription factor required for IFN2 expression. NSP1 targets IRF3 for proteasome degradation and down-regulates the IFN response. New data suggest NSP1 of a porcine rotavirus strain targets a different substrate and may inhibit activation of NF:B. Together, the data suggest rotavirus antagonizes multiple signaling molecules important in induction and effector functions of IFN. Systems development studies of this application will test the hypothesis that rotavirus inhibits IFN responses at multiple steps, and that inhibition is mediated primarily by NSP1. Our proteomics approach predicts that convergent signaling pathways will be revealed by quantitation of changes in relative protein abundance in rotavirus infected and NSP1-expressing cells. We will employ stable isotope labeling of amino acids in culture (SILAC) followed by high resolution chromatographic separation and tandem mass spectrometry. We will perform quantitative proteomic analyses of rotavirus infected cells in the context of the IFN response. SILAC technology will be used to label proteins in cells infected with rotavirus or treated with IFN and cell lysates will be analyzed by LC-tandem-MS. We will investigate mechanisms of NSP1-mediated resistance to IFN by defining target substrates and pathways affected by substrate inhibition. NSP1 will be expressed by a recombinant adenovirus in SILAC labeled cells. Changes in protein abundance will be analyzed to define NSP1 substrates and determine how these proteins integrate into antiviral networks.
Rotavirus infections are the major viral cause of acute vomiting and diarrhea in children under 5 years of age. These viruses are responsible for significant morbidity in developed countries with an estimated 2.7 million cases, ~600,000 doctor, outpatient, and emergency room visits, and 70,000 hospitalizations annually in the U.S. In developing countries, approximately 2 million children die of dehydrating diarrhea every year and nearly half of these are due to rotavirus infection. The initial cellular response to virus infection is induction of a specific pattern of gene expression regulated by interferon (IFN). Expression of IFN and IFN-regulated genes, if successful, result in establishment of an antiviral state that restricts virus replication and spread, while simultaneously initiating recruitment of the adaptive immune responses. Most, if not all viruses have evolved mechanisms to escape this response. Rotavirus protein NSP1 interferes specifically with activation of cellular protein IRF3 that is required for IFN expression. We recently discovered an additional mechanism by which NSP1 expression inhibits the IFN response. The studies described in this application propose a quantitative proteomic analysis of rotavirus infected cells and NSP1 expressing cells to determine changes in relative protein abundance potentially associated with induction of the IFN response. Understanding these pathways and the mechanisms by which viruses evade the innate immune response may lead new approaches to attenuated vaccines or antiviral drug targets specifically designed to bypass viral evasion strategies.
