Arkadeb Bhuinya, Vaccine and Infectious Disease Organization, University of Saskatchewan

Arkadeb Bhuinya1,2, Yusuke Kosugi8,9, Mohammad Golam Kibria8, Sophie-Marie Aicher1,5, Arianna M. Hurtado-Monzon1, Sauhard Shrivastava1,2 , Winfield Yim6, Kaushal Baid1, Ravendra Garg4, Shuhei Tsujino17,18, Takasuke Fukuhara17,18,19, Darryl Falzarano 1,2,3, Qiang Liu 1,2,4, Samira Mubareka5,6, Kei Sato8,9,10,11,12,13,14,15,16, Arinjay Banerjee 1,2,5,7 1Vaccine and Infectious Disease Organization, University of Saskatchewan, SK, Canada. 2Department of Veterinary Microbiology, University of Saskatchewan, SK, Canada. 3Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, SK, Canada. 4School of Public Health, University of Saskatchewan, SK, Canada. 5Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, ON, Canada. 6Sunnybrook Research Institute, Toronto, ON, Canada. 7Department of Biochemistry and Molecular Biology, University of British Columbia, BC, Canada. 8Division of Systems Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 9Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 10Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan. 11International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 12International Vaccine Design Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 13Collaboration Unit for Infection, Joint Research Center for Human Retrovirus infection, Kumamoto University, Kumamoto, Japan. 14MRC-University of Glasgow Centre for Virus Research, Glasgow, UK. 15Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. 16Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore. 17Department of Virology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan. 18Department of Microbiology and Immunology, Faculty of Medicine, Hokkaido University, Sapporo, Japan. 19Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, Japan.

Expressing & Multiplying

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the largest documented coronavirus pandemic. While SARS-CoV-2 accessory proteins are not essential for viral genome replication or transcription, they play critical roles in viral pathogenesis and immune evasion. Despite their importance, the regulatory mechanisms that fine-tune accessory protein expression at both the transcriptional and translational levels remain very poorly understood. In this study, we identify a virus-specific regulatory mechanism governing the expression of SARS-CoV-2 accessory gene, Orf6. Comparative genomic and functional analyses across 20 million SARS-CoV-2 sequences and known sarbecoviruses revealed that Orf6 generates markedly lower levels of sub-genomic RNA compared to other accessory genes. We confirmed this observation using three different sarbecoviruses (SARS-CoV-2, deer adapted SARS-CoV-2, and bat-derived BANAL-236) using human cellular models, hamsters, human clinical specimen, and deer and bat cell culture models. We discovered that the reduced transcript abundance of Orf6 is due to a missing nucleotide within the Orf6 viral promoter region. Surprisingly, despite its low sub-genomic RNA levels, Orf6 protein was produced at levels higher than other accessory proteins. These findings uncover a previously unrecognized dual-layer regulatory strategy in which non-coding viral elements decouple transcript abundance from protein expression. In addition, we demonstrate for the first time that heightened amounts of Orf6 protein in cells alters the cell cycle ultimately impacting overall cellular homeostasis. Our results highlight the importance of virus-specific promoter architecture in shaping coronavirus gene expression and pathogenicity using cutting-edge in vitro and in vivo models.