Coronavirus RNA synthesis by multicomponent protein machines

SUMMARY SARS-CoV-2, the causative agent of COVID-19, has emerged as a global human pathogen sweeping through our communities. The development of strategies to prevent or treat COVID-19 is essential for mitigating disease and limiting further viral spread. A key target of antiviral therapeutics is the virus RNA replication complex. The SARS-CoV-2 replication complex is a large multi-subunit machine with multiple co-factors, enzymes and host modulating proteins. How these protein subunits assemble and cooperate to carryout viral RNA replication and transcription remains unclear. The overarching theme of this work is to examine understudied aspects of the coronavirus replication complex with an emphasis on characterizing the effects of existing antiviral therapeutics as well as the discovery of novel targets and compounds. We are interested in how the virus nsp14 exonuclease contributes to viral RNA proofreading, reducing nucleotide misincorporations and providing natural resistance to nucleoside analogue drugs. We will also explore the function of the enigmatic nsp12 nucleotidyltransferase (NiRAN) and will piece together the network of viral protein interactions responsible for the assembly of the RNA synthesis complex. To this we will use diverse methods including biochemistry, biophysics, cell biology, chemical biology and cryo-electron microscopy. These studies will provide new insight into the workings of this complicated machine, provide new mechanisms of action for existing therapeutics and discover novel antiviral compounds. In addition, the high conservation of components of the replication machinery across the coronavirus family allows these studies of SARS-CoV-2 to be applicable to future emerging coronaviruses to head off future viral pandemics before they become global crises.