Roles for virus-derived circular RNAs during RNA virus infections

Stanford University, Stanford CA

Investigators

Abstract

Project Summary/Abstract Powerful direct-acting antivirals against hepatitis C virus (HCV) are available, but no vaccine is available. There are no effective antiviral compounds available to treat enterovirus 71 (RV71) that can cause severe neurological complications in children. In the case of SARS-CoV-2, powerful vaccines are available, but the rapid emergence viral variants warrant the continued development of novel antiviral approaches. Thus, it is important to continue to search for novel vulnerabilities in these RNA viruses. We have made the astonishing discovery that HCV-, EV71- and SARS-CoV-2-infected cells produce hundreds of virus-derived circRNAs (vcircRNAs) species of different sizes that are derived from the viral RNAs. Some of these vcircRNAs, such as the HCV internal ribosome binding site (IRES)-containing cluster I vcircRNA of HCV, are being translated to yield novel viral proteins with pro-viral functions. We also made the puzzling observation that the 3' end of the minus-strand of HCV contains an IRES that mediates the translation of a novel minus-strand protein (MSP) that is unique to the JFH1 type 2a genotype. Our hypothesis is that vcircRNAs modulate viral and cellular gene expression and function in infected cells to elicit novel pro-viral or anti-viral responses. The rationale for this proposal is that modulation of vcircRNA abundances affect the pathogenesis of HCV, EV71 and SARS-CoV-2. Our goals are to examine the functional roles for the vcircRNAs in human cultured liver cells, human liver organoids and lung A549-ACE2 cells, using bioinformatic, genetic, biochemical and cell biological approaches. Two specific aims are proposed to accomplish these goals. First, functional roles for abundant vcircRNAs and IRES-containing vcircRNAs in viral gene expression will be examined after depletion of vcircRNAs, using the Cas13-gRNA system, and after ectopic expression of in vitro-synthesized vcircRNAs. Analyses of intracellular vcircRNA localization and protein-vcircRNA interactions will be performed to gain mechanistic insights into the actions of vcircRNAs. The second aim we will test the hypothesis that the vcircRNAs are generated by cytoplasmic RNA ligases, such as the RTCB/archease ligase complex, that is involved in tRNA ligation and XBP1 mRNA splicing during the unfolded protein stress response, or the newly identified C12ORF29 ligase are involved in the formation of vcircRNAs. The expected outcomes from this application will address fundamental aspects about the functions of novel vcircRNAs and novel viral proteins in the viral infectious cycle. We hypothesize that other RNA virus families generate vcircRNAs as well, suggesting novel Achilles’ heels in viruses that can be targeted in novel antiviral approaches.