DESCRIPTION (provided by applicant): Viruses encode novel subsets of uncharacterized genes (predicted and hypothetical ORFs and noncoding RNAs) which can be expressed to modulate virus replication efficiency and/or host antiviral responses both in vitro and in vivo. Using highly pathogenic human respiratory and systemic viruses which cause acute and chronic life-threatening disease outcomes, we test the hypothesis that RNA and DNA viruses encode common and unique mechanisms to manipulate virus replication efficiency and host responses to determine severe disease outcomes. To address this hypothesis, the proposal takes advantage of novel expression vector platforms, synthetic gene design, reverse genetics, animal models of human disease, and a defined set of biochemical and immunologic assays to identify, characterize and then determine the role of uncharacterized genes in the lung (e.g., H5N1, SARS-CoV and human coronavirus EMC-1) and in systemic infections (e.g., Ebola and Human Herpes virus 8) both in vitro and in some instances, in vivo. Specifically, we test the hypothesis that these viral uncharacterized genes may function to auto-regulate virus replication efficiency, and/or function as an agonist or antagonize the host intracellular milieu to enhance virus replication, most likely be altering p53, innate immune sensing, inflammasome, apoptosis, and/or NF-?B signaling. To achieve these goals, a highly interactive group of experts in RNA and DNA virus pathogenesis and immunity work collectively to create a robust screening platform that rapidly identifies and characterizes the function of these uncharacterized genes in replication and pathogenesis. By identifying common key host bottleneck genes that are targeted by disparate virus pathogens, we identify rationale broadly relevant therapeutic targets for ameliorating disease outcomes in vivo. Importantly, this platform is: a) portable, b) can be rapidly applied to other highly pathogenic respiratory and microbial pathogens, c) will rapidly identify novel targets for therapeutic intervention, d) improve strategies for live attenuated or vectored virus vaccine design, and e) improve global responses to newly identified, epidemic disease outbreaks in human populations.

PUBLIC HEALTH RELEVANCE: H5N1, SARS-CoV, human coronavirus EMC-1, Ebola, and human herpesvirus 8 are on the list of NIAID Category A-C pathogens and Emerging/Re-Emerging pathogens. We propose to decipher the function of uncharacterized genes (unknown ORFs, hypothetical ORFs, non-coding RNAs) expressed by these RNA and DNA viruses in viral pathogenesis and evasion of host immunity. This will expand our understanding of the biology of these pathogenic human viruses and improve our ability to treat these viral infections. Project 1: Role of Uncharacterized Genes in High Pathogenic Human Coronavirus Infection Project Leader (PL): Baric, R. DESCRIPTION (provided by applicant): Coronaviruses encode six different human pathogens that target the upper and lower respiratory tract, resulting in mild to life-threatening disease. Two recently emerged human betacoronaviruses, the group 2b severe acute respiratory coronavirus (SARS-CoV) and the group 2c Human Betacoronavirus 2c EMC/2012 (HCoV SA1) emerged suddenly from bats to produce either a global outbreak or sporadic cases of severe acute respiratory distress syndrome with high mortality in human populations. While SARS-CoV is a category C select agent, both viruses encode a single-stranded positive polarity RNA genome of about 30,000 nucleotides in length, which encode for about 29 viral proteins, including several completely unique accessory ORFs that may contribute to in vivo pathogenesis. Importantly, coronaviruses encode a variety of unknown and hypothetical ORFs as well as noncoding RNAs and miRNAs. We test the hypothesis that subsets of these novel genes play critical roles in regulating virus replication efficiency and in vivo pathogenesis. To address this hypothesis, the proposal takes advantage of novel expression vector platforms, synthetic gene design, reverse genetics, animal models of human disease, and a defined set of biochemical and immunologic assays to identify, characterize and then determine their role in SARS-CoV, EMC-1 and select ancestral bat coronaviruses in vitro and in vivo. Specifically, we test the hypothesis that these viral genes function to antagonize/modulate the host intracellular milieu to enhance virus replication, most likely be altering p53, innate immune sensing, inflammasome, apoptosis or TLR signaling. In Aim 1, we characterize the expression, subcellular localization and function as an antagonist or agonist of antiviral defense signaling networks. In Aim 2, we isolate recombinant virus and study the function of these uncharacterized genes in virus replication and host cell antiviral defense signaling networks. In Aim 3, we study the pathogenesis of recombinant viruses lacking unknown and hypothetical ORFs and noncoding RNAs using young and aged animal models of human disease. RELEVANCE: Coronaviruses are important human pathogens and encode novel genes that antagonize host antiviral defense pathways. By identifying the mechanisms of antagonism and the host targets, our studies will reveal novel therapeutic targets for disease control and new strategies to attenuate coronaviruses, protecting the global health from future outbreaks of disease.