The RNA demethylase FTO controls m6A marking on SARS-CoV-2 and classifies COVID-19 severity in patients (preprint)

The RNA modification N6-methyladenosine (m6A) plays a key role in the life cycles of several RNA viruses. Whether this applies to SARS-CoV-2 and whether m6A affects the outcome of COVID-19 disease is still poorly explored. Here we report that the RNA demethylase FTO strongly affects both m6A marking of SARS-CoV-2 and COVID-19 severity. By m6A profiling of SARS-CoV-2, we confirmed in infected cultured cells and showed for the first time in vivo in hamsters that the regions encoding TRS_L and the nucleocapsid protein are multiply marked by m6A, preferentially within RRACH motifs that are specific to {beta}-coronaviruses and well conserved across SARS-CoV-2 variants. In cells, downregulation of the m6A demethylase FTO, occurring upon SARS-CoV-2 infection, increased m6A marking of SARS-CoV-2 RNA and slightly promoted viral replication. In COVID-19 patients, a negative correlation was found between FTO expression and both SARS-CoV-2 expression and disease severity. FTO emerged as a classifier of disease severity and hence a potential stratifier of COVID-19 patients.

Betulonic acid derivatives inhibiting coronavirus replication in cell culture via the nsp15 endoribonuclease (preprint)

The lack of medication to suppress coronavirus infections is a main reason for the dramatic course of the COVID-19 pandemic. There is an urgent need to identify suitable coronavirus drug targets and corresponding lead molecules. Here we describe the discovery of a class of coronavirus inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. SAR exploration of these 1,2,3-triazolo fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 M) of human coronavirus 229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

Human coronaviruses disassemble processing bodies (preprint)

The Coronaviridae are a family of viruses with large RNA genomes. Seven coronaviruses (CoVs) have been shown to infect humans, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease of 2019 (COVID-19). The host response to CoV infection is complex and regulated, in part, by intracellular antiviral signaling pathways triggered in the first cells that are infected. Emerging evidence suggests that CoVs hijack these antiviral responses to reshape the production of interferons and proinflammatory cytokines. Processing bodies (PBs) are membraneless ribonucleoprotein granules that mediate decay or translational suppression of cellular mRNAs; this is particularly relevant for proinflammatory cytokine mRNA which normally reside in PBs and are repressed. Emerging evidence also suggests that PBs or their components play important direct-acting antiviral roles, providing a compelling reason for their frequent disassembly by many viruses. No information is known about how human CoVs impact PBs. Here, we provide data to show that infection with the human CoV, OC43, causes PB disassembly. Moreover, we show that several SARS-CoV-2 gene products also mediate PB loss and virus-induced PB loss correlates with elevated levels of proinflammatory cytokine mRNAs that would normally be repressed in PBs. Finally, we demonstrate that stimulating PB formation prior to OC43 infection restricts viral replication. These data suggest that SARS-CoV-2 and other CoVs disassemble PBs during infection to support viral replication and evade innate immune responses. As an unintended side effect, the disassembly of PBs enhances translation of proinflammatory cytokine mRNAs which normally reside in PBs, thereby reshaping the subsequent immune response.

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways (preprint)

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33{degrees}C instead of 37{degrees}C. Even stronger preference for the upper airway temperature of 33{degrees}C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37{degrees}C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37{degrees}C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology. SIGNIFICANCE STATEMENTThe rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33{degrees}C) or lower (37{degrees}C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.