#25 The highly conserved stem-loop II RNA element (s2m) is critical for the lifecycle of astrovirus VA1 but is dispensable for SARS-CoV-2

Abstract Background RNA viruses often contain important elements for the viral lifecycle in the untranslated regions (UTR) of the genome. The stem-loop II (s2m) element is a RNA structural motif in the 3’ UTR that is 39-43 nucleotides in length and is present in members of the Astroviridae, Caliciviridae, Picornaviridae, Reoviridae, and Coronaviridae viral families, including SARS coronavirus 1 and 2 (SARS-CoV-1 and -2). Despite the first description of this element twenty-five years ago, the functional significance of the s2m sequence to the viral lifecycle remains poorly understood. The conservation of the s2m in many viral genomes suggests some fitness benefit to viruses that maintain it. Method To test the significance of the s2m element, we developed reverse genetic systems for astrovirus VA1 (VA1) and SARS-CoV-2. For both viruses, we introduced deletions and mutations predicted to significantly disrupt the secondary structure of the s2m. Recombinant viruses were passaged and the infectious viral titer was measured. To evaluate the in vivo significance of the s2m, we used a hamster model of SARS-CoV-2 infection. Recombinant viruses were intranasally inoculated and viral RNA and infectious viral titers measured from the nasal washes and the lungs three and six days after infection. A clinical SARS-CoV-2 isolate containing a deletion of the s2m was also propagated and the viral titer measured in a multi-step growth curve. Results The s2m in VA1 was critical to the viral lifecycle as deletion of the s2m resulted in recombinant virus that could not be rescued and propagated. Introduction of one or two nucleotide mutations that disrupt predicted G-C binding pairs in the s2m element also resulted in recombinant virus that was not viable. Compensatory mutations that restored G-C base pairings in the s2m element supported recovery and propagation of the virus with similar viral titers compared to wild-type (WT) VA-1. In contrast, the s2m in SARS-CoV-2 was dispensable. SARS-CoV-2 genomes containing a deletion or mutation of the s2m could be rescued and propagated in cell culture and had similar viral titers compared to the WT SARS-CoV-2 in multi-step growth curves. In the hamster model, there was also no difference in the amount of viral RNA and infectious virus in the lung and nasal washes of infected animals. We also identified a clinical SARS-CoV-2 isolate that contained a deletion of the s2m and it showed no differences in the growth in a multi-step growth curve compared to a SARS-CoV-2 virus with an intact s2m. Conclusion The s2m is essential for the lifecycle of astrovirus VA1 but dispensable for SARS-CoV-2, suggesting differing functions of the s2m that is virus dependent. Future experiments will identify the mechanistic basis for the divergent role of the s2m in each viral lifecycle.