Modulation of Middle East respiratory syndrome coronavirus infection by human USP13 protein

Abstract

Two major outbreaks of highly pathogenic coronaviruses, Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV-1) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), have been reported in 2003 and 2012 respectively. Most recently, a newly identified coronavirus, SARS-CoV-2, has emerged and causes more than 100 million human infection cases and two million deaths globally (as of March 2021). However, no clinically proven effective antiviral drug is available for the treatment of highly pathogenic coronavirus infections. There are two classes of antivirals: one targets the viral components such as polymerases or helicases, while another targets the host cellular factors that support the virus infection. To discover the host-targeting antivirals, the first step is to identify the key host factors or cellular pathways that support virus replication. In this study, the host factors or pathways were identified by the host-MERS-CoV interactomes study. More than 700 host factors that associate 25 MERS-CoV proteins were identified. Six of them (USP13, SIRT1, GSK3B, PTGES2, TTK and TPP2) were selected and further confirmed by immunoprecipitation. Commercially available small chemical inhibitors targeting these six factors or their corresponding pathways were used for further evaluation of antiviral efficacy against MERS-CoV. As expected, all these inhibitors potently suppressed MERS-CoV in human lung epithelial Calu-3 cells and human embryonic kidney 293 cells stably expressing DPP4, a receptor for MERS-CoV entry. It found that USP13 inhibitor showed the strongest inhibitory effect against MERS-CoV infection. USP13 is known as a deubiquitinase that can stabilize the host protein such as STAT1, a key transcription factor for type I IFN signaling. It was demonstrated that USP13 directly bound to MERS-CoV NSP16, and hence augment the NSP16 protein expression. This effect requires the ubiquitin-binding domain of USP13, but not depends on its catalytic domain. Lysine mapping showed that NSP16 lysine 249 and 253 were required for this enhancement of protein expression by USP13. Since the non-structural genes are conserved among different coronaviruses, it showed that USP13 also strongly enhanced the expression of NSP16 proteins derived from other five human coronaviruses (SARS-CoV-2, SARS-CoV-1, HCoV-229E, HCoV-NL63 and HCoV-OC43). Besides, USP13 specific inhibitor, Spautin-1, potently suppressed the three highly pathogenic human coronaviruses (MERS-CoV, SARS-CoV-1 and SARS-CoV-2) infection in vitro, suggested that USP13 is a potential host dependency factor for human coronavirus infection. Encouragingly, Spautin-1 also exhibited the same antiviral activity in SARS-CoV-2 infected human ex-vivo lung tissues. USP13 is the upstream deubiquitinase of Beclin-1/Vps34 complex that plays a key role on the omegasome formation. Therefore, USP13 may promote coronavirus infection on top of stabilizing the viral NSP16 protein. Indeed, it found that the Vps34 inhibitor (VPS34-IN1) strongly suppressed SARS-CoV-2 infection in both cultured cells and human ex-vivo lung tissues. Taken together, USP13 acts on both viral and host targets to promote human coronavirus infection. Thus, USP13 and its signaling pathways will be a good druggable target for the development of antivirals against highly pathogenic human coronaviruses including the recently emerged SARS-CoV-2. (An abstract of 481 words)