RNA Recombination in Coronaviruses

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RNA viruses are exceptionally diverse and rapidly evolving. Their RNA-dependent RNA polymerases are prone to mutation, lack proof-reading capabilities (with the unique exception of coronaviruses) and frequently undergo recombination between both homologous and non-homologous templates. This confers the ability to rapidly adapt to new environments, evade immune responses and side-step anti-viral therapies. In many RNA viruses, RNA recombination is co-regulated with replication fidelity and is required to correct deleterious mutations and thus is a critical determinant of viral fitness. Through the combined generation subgenomic messenger RNAs, structural variants (SVs), and Defective RNAs (D-RNAs), RNA recombination is an essential property of CoV replication and evolution. With the continued spread of SARS-CoV-2, recombination has been highlighted as a major factor driving the emergence of novel variants. SARS-CoV-2 variants have developed mutations thought to improve receptor binding, disrupt innate immune responses, or evade adaptive immunity. We recently demonstrated that SARS-CoV-2 is >10-fold more recombinogenic than other CoVs such as MERS and MHV (Gribble et al, 2021, PLoS Path). Interestingly, RNA recombination events that give rise to SVs and D- RNAs were predominantly found adjacent to U-rich tracts. Using ‘Tiled-ClickSeq’, developed in our lab (Jaworski et al, 2021, eLife), we found the same trends in COVID19 patient samples. We also demonstrated that micro- deletions flanked by U-rich motifs in the Spike protein of SARS-CoV-2 spontaneously arise during passaging in cell culture and alter viral pathogenesis (Johnson et al, 2021, Nature). Notably, microindels in novel variants of SARS-CoV-2 (e.g. Alpha variant) are also flanked by U-rich motifs. Altogether, this evidence suggests U-rich tracts define RNA recombination hotspots which have given rise to the emergence of novel CoVs variants. However, while recombination rates are demonstrably high, SARS-CoV-2 has a strong transmission bottleneck restricting the dissemination of minority variants. Novel variants such as Alpha and Omicron contain multiple recombination events and may have arisen during intrahost adaption in a chronically infected (e.g. immunocompromised) patient. Therefore, characterizing RNA recombination in the correct physiological setting is crucial to understand which RNA recombinant species are able to emerge and how these are selected. In this proposal, we will characterize the molecular mechanisms of RNA recombination and characterize how the physiological aspects and site of infection determines whether a novel variant is selected for and thus emergence. Characterizing the factors that give rise to new emergent strains and variants will be critical in our understanding of both historical and future outbreaks events. Furthermore, these studies will inform on the basic and fundamental principles that drive RNA virus evolution. The current pandemic presents a unique situation in which to characterize the basic principles of virus emergence and may impact our understanding of a range of viral pathogens.