Mechanism-based Targeting of the RNA Processing Machinery of SARS-CoV-2

Modified Project Summary/Abstract Section The massive global pandemic with high morbidity and mortality makes Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) one of the deadliest viruses in recent history. It is especially noteworthy for hijacking the normal operations of human cells. To develop effective therapies, we need a better understanding of the mechanisms that permit the virus to invade cells and evade host immune restriction. SARS-CoV-2 encodes the non-structural protein (nsp)16/nsp10 protein complex that transfers a methyl group from S-adenosyl methionine (SAM) to 2'-OH of the first transcribing nucleotide of the viral mRNA and thus converts the Cap-0 (m7GpppA) to Cap-1 (m7GpppAm). The resulting viral mRNA mimics host cell's mRNA. In this way, a cell cannot distinguish between its own RNA and that of the virus. This modification of the virally encoded mRNA not only tricks the immune system and helps the virus to take over the host translation machinery for synthesis of its own proteins for survival and propagation. Ablation of nsp16 activity should trigger an immune response to viral infection and limit pathogenesis. Our recent paper in Nature Communications described atomic level details of the nsp16/nsp10 complex and how the enzyme is well adapted to bind the RNA cap and exert the 2'-OH methylation. We also discovered a distant pocket (located 25Å away from the catalytic center) in nsp16 that is unique to SARS-CoV-2. We also found that this pocket in nsp16 is partially composed of amino acids that are unique to SARS-CoV- 2. It can bind small molecules outside of the catalytic center. We propose to build a long-term research program aimed at deciphering the factors crucial to the maintenance of RNA genome and evasion from the host's immune response. Our studies will reveal basic principles underlying SARS-CoV-2 RNA cap modification, the mode of nucleoprotein (NP) assembly, interplay with mRNA, and new approaches for therapeutic targeting. In Aim 1, we will resolve a series of new structures of nsp16/nsp10 proteins captured in every step of the methyl transfer by X-ray crystallography. The structural data will be validated by detailed biochemical and biophysical studies. We will resolve the biochemical and structural determinants of the assembly of viral RNA capping machinery, and identify factors underlying integrity of RNA genome. In Aim 2, we will develop a novel molecular tool to study temporal distribution of the RNA methylation during viral infection. We will examine new models for combinatorial inhibition of viral proteins by drug repurposing or novel small molecules. These small molecules will also be tested against other viruses, such as Ebola, Lassa, and MPox, for their broad utility as pan-antivirals. Finally, we will use our recently established reverse genetics approaches based on the use of a bacterial artificial chromosome (BAC) to generate recombinant (r)SARS-CoV2 containing mutations in nsp16 to determine their contribution in viral replication in cultured cells and pathogenesis in vivo using our recently described K18 human angiotensin converting enzyme 2 (hACE2) mouse model of SARS-CoV-2 infection and associated coronavirus disease 2019 (COVID-19).