Uncovering the mechanism of RNA virus inactivation by UV-C irradiation and Coronavirus immune evasion via TonEBP cleavage

Abstract

In the 21st century, RNA viruses have been responsible for outbreaks of two global pandemics. H1N1 influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused H1N1 flu pandemic in 2009 and COVID-19 pandemic in 2019 respectively. Respiratory tract infection with H1N1 or SARS-CoV-2 results in widespread clinical disease severity in humans. Both the Influenza virus and coronavirus are zoonotic RNA viruses that are rapidly evolving and highly evasive to vaccines. UV-C irradiation is considered an efficient tool to reduce the risk of viral transmission of H1N1 and SARS-CoV-2 regardless of their evolution. Although UV-C is known to inactivate these RNA viruses, the underlying mechanisms are not completely understood. Therefore, I the first aim of this study was to elucidate whether the inactivation of RNA viruses by UV-C is dependent on genomic damage. Additionally, coronavirus has diverse strategies to evade host immune responses by hijacking host proteins. Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, is a versatile stress protein associated with a range of immunometabolic disorders. While the antiviral or proviral functions of TonEBP in respective coxsackievirus B3 (CVB3) or lymphocytic choriomeningitis virus (LCMV) infection have been well studied, the role of TonEBP in coronavirus infection remains unknown. Thus, the second aim of this study was to investigate the function of TonEBP in coronavirus immune evasion. Here I first show that UV-C irradiation efficiently inactivates H1N1 and two human coronaviruses, HCoV-OC43 and HCoV-229E as surrogates for SARS-CoV-2. Interestingly, the inactivation rates of these ssRNA viruses are proportional to their genome size. Based on long-range reverse transcription followed by quantitative polymerase chain reaction (qPCR), I predicted the regression rate of undamaged genome by extrapolating the experimentally observed RNA damage on different-sizedamplicons to the whole genome size. A comparison of the predicted regression rate of intact genome to the inactivation rate revealed that UV-C induced inactivation of RNA viruses is mainly due to viral genome damage. Secondly, I show that coronavirus non-structural protein 5 (NSP5) cleaves TonEBP and suppresses IFN-β induction to evade the host immune response. Coronavirus infection induces a cleavage of TonEBP by NSP5 targeting Q1127 amino acid of TonEBP. The cleaved C-terminal fragment of TonEBP is removed by proteasomal degradation. The cleaved N-terminal fragment of TonEBP (TonEBP NT, i.e., TonEBP 1-1127) inhibits IFN-β induction to establish productive coronavirus infection. A comparison of coronavirus propagation in U2OS stable cells expressing either TonEBP wild-type or TonEBP Q1127A mutant resistant to cleavage by NSP5 revealed that TonEBP cleavage enhances coronavirus replication. Notably, TonEBP NT interacts with p65 and directly binds to IFN-β promoter region, which indicates that TonEBP NT inhibits IFN-β induction by replacing p65 bound to IFN-β promoter. In conclusion, UV-C is an efficient tool for disinfecting RNA viruses including influenza and coronavirus by inducing viral genome damage, and coronavirus evades host immune response by cleaving TonEBP to suppress IFN-β induction.