Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

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Abstract
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. Viruses that replicate in the host cell cytoplasm have evolved to employ host cell-derived membranes to compartmentalize genome replication and transcription. Specifically for positive-stranded RNA viruses, accumulating knowledge concerning the involvement, rearrangement and requirement of cellular membranes for RNA synthesis specify the establishment of the viral replicase complex at host cell-derived membranes as an evolutionary conserved and essential step in the early phase of the viral life cycle. Here we describe a small compound inhibitor of coronavirus replication that (i) specifically targets this membrane-bound RNA replication step and (ii) has broad antiviral activity against number of diverse coronaviruses including highly pathogenic SARS-CoV and MERS-CoV. Since resistance mutations appear in an integral membrane-spanning component of the coronavirus replicase complex, and since all positive stranded RNA viruses have very similar membrane-spanning or membrane-associated replicase components implicated in anchoring the viral replication complex to host cell-derived membranes, our data suggest that the membrane-bound replication step of the viral life cycle is a novel, vulnerable, and druggable target for antiviral intervention of a wide range of RNA virus infections.