Date Published: May 29, 2014
Publisher: Public Library of Science
Author(s): Anna Lundin, Ronald Dijkman, Tomas Bergström, Nina Kann, Beata Adamiak, Charles Hannoun, Eveline Kindler, Hulda R. Jónsdóttir, Doreen Muth, Joeri Kint, Maria Forlenza, Marcel A. Müller, Christian Drosten, Volker Thiel, Edward Trybala, Andrew Pekosz.
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.
Prior to the emergence of the highly pathogenic severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in 2003 – only two circulating human coronaviruses (HCoVs), HCoV-229E  and HCoV-OC43  causing relatively mild common cold-like respiratory tract infections, were known, and coronaviruses have not been regarded as significant threat for human health. Now, more than ten years later, the emergence of another highly pathogenic coronavirus of zoonotic origin, the Middle East respiratory syndrome coronavirus (MERS-CoV) –, boosted community awareness towards the pending need to develop effective therapeutic options to combat coronavirus infections.
Here we describe the discovery of a novel class of inhibitor and propose a mode-of-action that targets membrane-bound viral replication. Like all positive strand RNA viruses, coronaviruses employ host cell membranes to assemble the viral replicase complex. This evolutionary conserved strategy provides a compartment for viral RNA synthesis that is enriched in replicative viral and host cell-derived proteins and believed to protect from antiviral host cell defense mechanisms. The remarkable efficacy of K22-mediated inhibition of coronavirus replication confirms that the employment of host cell membranes for viral RNA synthesis is a crucial step in the coronavirus life cycle, and importantly, demonstrates that this step is extremely vulnerable and also druggable for antiviral intervention.