Constructing the nest - understanding the mechanisms of nidoviridae RNA genomes transcription and recombination

PROJECT SUMMARY Coronaviruses belong to the higher order of Nidovirales, which are enveloped positive sense, single stranded RNA viruses that infect vertebrates and invertebrates. Nidoviruses are named from the Latin "nidus", meaning nest as viruses in this order produce multiple distinct genomic transcripts with common ends during infection. These transcripts, which express all of the viral structural proteins, are shorter than the full-length genome and are termed subgenomic RNAs. Subgenomic RNAs are generated through a process called discontinuous transcription, in which the viral RNA polymerase dissociates from its template midway through transcription and finishes transcribing on a different template. Despite nearly half a century of research on "nested" viruses, we have limited knowledge of the host proteins, the RNA template features, and the mechanisms involved in discontinuous transcription. Moreover, the same principles and players governing production of subgenomic RNAs are thought to be involved in recombination of coronaviruses, in which two distinct parental viruses coinfect the same host cell and generate virus progeny that have genes from both parents. Recombination is at the heart of viral evolution and is critical for the ability of a virus to adapt to new hosts. Understanding the process of coronavirus discontinuous transcription and recombination will open new avenues for therapeutic intervention. My overall goal is to use high throughput and unbiased approaches to identify the RNA features, including sequence and structure, as well as the protein factors that are necessary for subgenomic RNA production and viral recombination for SARS-CoV-2. I will build upon my previous work on developing next generation sequencing approaches to study the cis features of viral RNAs such as SARS-CoV-2 during active infection. I will establish new assays to study viral recombination and use CRISPR screens to identify the host trans factors required for these processes. The proposed project will address fundamental questions of coronavirus biology. Amongst these questions are the SARS-CoV-2 recombination frequency, factors that promote recombination, and the existence of recombination "hot spots". The answers to these questions will uncover viral vulnerabilities and aid our understanding of coronavirus evolution, all which will help prevent future pandemics.