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

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.

Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus

“…2; Supplemental Table S2). Nsp6 is a membrane-spanning protein and is an integral component of the viral replication complex involved in double-membrane vesicle formation (Lundin et al 2014). Although the functional importance of these MERS-CoV variants in viral replication and their interaction with the host immune system remain to be elucidated, our data showed that the selection of these variants may not be independent from each other, suggesting the unit of selection may be a combination of variants rather than individual variants.…”

Structure-activity relationship studies of lipophilic teicoplanin pseudoaglycon derivatives as new anti-influenza virus agents

“…To perform the qRT-PCR-based coronavirus yield assay, the materials were: HCoV-229E nucleoprotein-derived forward primer (5 0 -TTAGAGAGCGTGTTGAAGGTG-3 0 ); reverse primer (5 0 -GTTCTGAATTCTTGCGCCTAAC-3 0 ); probe (5 0 -6-FAM-TCTGGGTTG/ ZEN/CTGTTGATGGTGCTA-IBFQ-3 0 ; and a standardization plasmid with a 294-bp sequence of the HCoV-229E N-gene. As reference compound, the HCoV RNA synthesis inhibitor K22 [35] (from ChemDiv) was used.…”

Middle East respiratory syndrome coronavirus: current knowledge and future considerations

“…Promising treatment approaches currently emerging in the scientific literature (66) include the novel molecule K22, which targets viral replication without cellular toxicity (99). It targets viral MERS-CoV 3C protease (79) and the interface between the MERS-CoV receptor-binding domain (RBD) and the receptor by inhibiting the enzymatic function of dipeptidyl peptidase 4 (DPP4) (100).…”