Unheeded SARS-CoV-2 protein? Look deep into negative-sense RNA 1 Martin Bartas 1, *, # , Adriana Volná 2, # , Jiří Červeň 1 , Václav Brázda 3 & Petr Pečinka 1, * 2 3 1 Department of Biology and Ecology, University of Ostrava, Ostrava, 710 00, Czech Republic 4 2 Department of Physics, Faculty of Science, University of Ostrava, Ostrava, 71000, Czech Republic 5 3 Institute of Biophysics, Czech Academy of Sciences, Brno, 612 65, Czech Republic 6 * Correspondence and requests for materials should be addressed to Martin Bartas 7 (email: martin.bartas@osu.cz) or Petr Pečinka (email: petr.pecinka@osu.cz) 8 # These authors contributed equally to this work 9 10 11 Abstract: 12 SARS-CoV-2 is a novel ssRNA+ virus from the Coronaviridae family, which has caused the 13 COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest of RNA viruses, 14 comprising of 26 known protein-coding loci. This study aimed to explore the coding potential 15 of negative-strand RNA intermediate for its potential to contain additional protein coding-loci. 16 Surprisingly, we have found several putative ORFs and one brandt new functional SARS-CoV- 17 2 protein-coding loci and called it Avo1 (Ambient viral ORF1). This sequence is located on 18 negative-sense RNA intermediate and bona fide coding for 81 amino acid residues long protein 19 and contains a strong Kozak sequence for translation on eukaryotic ribosomes. 20 In silico translated protein Avo1 has a predominantly alpha-helical structure. The existence of 21 the Avo1 gene is supported also by its evolutionarily and structural conservation in RaTG13 bat 22 coronavirus. The nucleotide sequence of Avo1 also contains a unique SREBP2 binding site 23 which is closely related to the so-called “cytokine storm” in severe COVID-19 patients. 24 Altogether, our results suggest the existence of still undescribed SARS-CoV-2 protein, which 25 may play an important role in the viral lifecycle and COVID-19 pathogenesis. 26 27 28 29 30 31 . CC-BY-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 15, 2020. ; https://doi.org/10.1101/2020.11.27.400788 doi: bioRxiv preprint