Novel Nucleic Acid-Binding Proteins in Emerging Viruses

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Abstract SARS and MERS are human respiratory coronaviruses with pandemic potential. The MERS virus affects 25 countries worldwide and has a case fatality rate of 30-40%. There is no effective drug or vaccine against a coronavirus. Only a few coronavirus proteins (10-35%) have solved structures, making it difficult to understand viral protein interactions with binding partners. The long-term goal of our research is to define the structural biochemistry of unusual nucleic acid structures, including poly(ADP-ribose), and the viral proteins that recognize and process these biomolecules. Analysis of the coronavirus (CoV) genome shows that many coronavirus proteins have potential nucleic acid binding and processing functions. Guanine quadruplexes contribute to transcriptional and translational regulation of viral genes. This regulation allows the virus to evade the immune system and control viral gene expression. The roles of guanine quadruplex-binding proteins are just beginning to be recognized, and the structural basis of guanine quadruplex binding is poorly understood. Another unusual nucleic acid, poly(ADP-ribose), is a post-translational modification that regulates more than 20 biochemical pathways. PAR and PAR-synthesizing enzymes contribute to antiviral activity and virus-host interactions. There is presently no knowledge of the effect of this PTM on peptides and proteins, and little knowledge of the conformation of PAR itself. Our research employs solution NMR, biochemical and computational techniques to investigate these viral proteins. The central hypothesis of our work is that both guanine quadruplex-binding and PAR-binding macrodomain proteins occur in the nonstructural proteins of coronaviruses. Our work will develop structure-function relationships for these proteins, many of which are highly divergent relative to known proteins. We focus on emerging viruses such as MERS, SARS, and bat coronaviruses, which are primary animal reservoirs for coronavirus evolution. We will use gel shift assays, enzyme assays and SELEX to identify viral and host sequences that are targets of viral protein interaction. We will use large-scale biochemical screens to identify other biochemical functions for these proteins. In addition, we will examine conformations of the unusual nucleic acid PAR and determine the conformational effects of PARylation on proteins using molecular simulations, rapid acquisition NMR, and enzymatic synthesis to produce new isotope-labeled PAR oligomers. Conformational changes upon PARylation may contribute to enzymatic activation or inhibition, and have important implications for the design of inhibitors and antivirals.