Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Coronaviruses from both the Alphacoronavirus and Betacoronavirus genera interfere with the type I interferon (IFN) response in various ways, ensuring the limited activation of the IFN response in most cell types. Of the gammacoronaviruses that mainly infect birds, little is known about the activation of the host immune response. We show that the prototypical Gammacoronavirus, infectious bronchitis virus (IBV), induces a delayed activation of the IFN response in primary renal cells, tracheal epithelial cells, and a chicken cell line. In fact, Ifn␤ expression is delayed with respect to the peak of viral replication and the accompanying accumulation of double-stranded RNA (dsRNA). In addition, we demonstrate that MDA5 is the primary sensor for Gammacoronavirus infections in chicken cells. Furthermore, we provide evidence that accessory proteins 3a and 3b of IBV modulate the response at the transcriptional and translational levels. Finally, we show that, despite the lack of activation of the IFN response during the early phase of IBV infection, the signaling of nonself dsRNA through both MDA5 and TLR3 remains intact in IBVinfected cells. Taken together, this study provides the first comprehensive analysis of host-virus interactions of a Gammacoronavirus with avian innate immune responses. IMPORTANCEOur results demonstrate that IBV has evolved multiple strategies to avoid the activation of the type I interferon response. Taken together, the present study closes a gap in the understanding of host-IBV interaction and paves the way for further characterization of the mechanisms underlying immune evasion strategies as well as the pathogenesis of gammacoronaviruses. Coronaviruses constitute a large family of positive-stranded RNA viruses and cause a range of human and veterinary diseases. Infectious bronchitis virus (IBV) is the prototype avian coronavirus from the Gammacoronavirus genus and the causative agent of a highly contagious respiratory disease of major economic importance to the poultry industry (1). IBV enters the avian host through the respiratory tract, where it causes the destruction of the epithelium, leading to respiratory distress and initiation of secondary bacterial infections. Depending on the strain, IBV also can spread to other epithelial surfaces, such as the gastrointestinal tract, the kidneys, and the oviduct, with the latter causing problems in egg production and quality (1-6). Contrary to coronaviruses from the Alphacoronavirus and Betacoronavirus genera, including human coronavirus HCoV-229E, severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV), and mouse hepatitis virus (MHV), very little is known about how gammacoronaviruses, including IBV, evade or interfere with the innate immune responses of their host.Innate immune responses consist of a network of antimicrobial mechanisms, of which the type I interferon (IFN) response is an essential defense mechanism against viruses. Typically, the type I IFN response, here referred to as the IFN response, is...