Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential

The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoVEMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoVEMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. Importance A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

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 recombinant HCoV-229E nsp6 mutants.

<p>(<b>A</b>) Predicted topological structure of HCoV-229E nsp6 indicating the location of K22 resistance mutations. Concerning transmembrane domains VI and VII two proposed topologies are shown. (<b>B-C</b>) Recombinant nsp6 mutant viruses are resistant to K22. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) was added at specific time points relative to the end of inoculation period. The infectious cell culture medium and cells were harvested after 24 h of incubation at 37°C, and copy numbers of cell-associated (CA) or extracellular (EX) viral RNA was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. (<b>D-F</b>) Replication kinetics of recombinant nsp6 mutant viruses. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at an moi of 0.05 for 1 h at 4°C. The infectious cell culture medium and cells were harvested at specific time points relative to the end of inoculation period, and copy numbers of cell-associated (CA; <b>D</b>) or extracellular (EX; <b>E</b>) viral RNA and infectivity (<b>F</b>) was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments.</p

K22 structure, antiviral activity, and cytotoxicity.

<p>(<b>A</b>) K22 structure. (<b>B</b>) Anti-HCoV-229E activity of K22 in MRC-5 cells. K22 and HCoV-229E were added to MRC-5 cells, and the number of viral plaques developed after 48 h were assessed. Data shown are means (±SD) of duplicate determinations from three independent experiments. PFU, plaque forming unit. (<b>C</b>) Viability and proliferation of MRC-5 cells in the presence of K22. MRC-5 cells were incubated with K22 or DMSO solvent for 48 h at 37°C and the cell viability determined using tetrazolium-based reagent while cell proliferation was assayed by counting of cells. Data shown are means (±SD) of duplicate determinations from two independent experiments. (<b>D</b>) K22 affects the post-entry phase of viral life cycle. K22 (4 µM) or DMSO solvent were incubated with cells for a period of 2 h either before (−2 h), during (0 h) or after a 2 h period of cell inoculation with HCoV-229E, and the number of viral plaques developed after 48 h were assessed. Data shown are means of duplicate determinations from three independent experiments.*<i>P</i><0.05; <i>n</i> = 3. ***<i>P</i><0.005; <i>n</i> = 3. (<b>E-F</b>) K22 exhibits potent antiviral activity when added up to 6 h after infection of cells. MRC-5 cells were inoculated with HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) added at specific time points relative to the end of inoculation period. The culture medium and cells were harvested after 24 h of incubation at 37°C, and the viral RNA (<b>E</b>) and infectivity (<b>F</b>) determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. EX, extracellular medium; CA, cell-associated sample.</p

Alterations detected in the K22 resistant variants of HCoV-229E.

a<p>Detected by comparison of the nucleotide sequences of HCoV-229E subjected to 10–13 passages in the presence of K22 including its plaque purified variants A-R with those of initial virus or mock-passaged virus (accession number KF293665).</p>b<p>Plaque purified HCoV-229E that served as initial material for the virus passages.</p>c<p>IC50 (µM).</p>d<p>Fold resistance to K22 as related to initial virus is shown in parentheses.</p>e<p>Virus preparation and its plaque purified variants M-R obtained in separate K22 selection experiment.</p>f<p>The virus used for preparation of recombinant nsp6 mutants.</p>g<p>K22 resistant recombinant viruses.</p

Alignment of coronavirus nsp6 sequences.

<p>Alignment of nsp6 sequences derived from coronaviruses used in this study was performed with Geneious Software (Biomatters Ltd, New Zealand). Coronavirus species and corresponding GenBank accession numbers are indicated. Membrane domains predicted by TMHMM Server v. 2.0 (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>) are indicated by cyan shading while conserved amino acid residues are highlighted by black/grey shading. K22 resistance-conferring mutations in HCoV-229E nsp6, identified in this study, are depicted.</p

K22 affects formation of double membrane vesicles (DMVs).

<p>MRC-5 cells growing on Melinex polyester film were infected with wild type HCoV-229E (WT) or with K22-resistant recombinant nsp6 mutant HCoV-229E^M159V (M159V) and incubated for 18 h at 37°C with or without K22. The cells were then fixed with glutaraldehyde and processed for electron microscopy without their scrapping or pelleting. (<b>A</b>) Electron micrographs of cells infected with WT virus show presence of perinuclear clusters of DMVs (arrow) and viral particles (arrowhead), and the lack of their production upon K22 treatment (4 µM). (<b>B</b>) Note presence of DMVs and viral particles in cells infected with K22-resistant nsp6 recombinant HCoV-229E^M159V (M159V) irrespective of the addition of K22. Each image shown was selected from a pool of over 30 images captured in three separate experiments.</p

K22 affects formation of coronavirus replication complex in cells.

<p>MRC-5 cells were infected with wild type HCoV-229E (WT) and K22-resistant recombinants HCoV-229E^H121L (H121L), HCoV-229E^M159V (M159V), and HCoV-229E^H121L/M159V (H121L/M159V) and incubated for 18 h with or without the presence of K22. The cells were then fixed with 4% paraformaldehyde and immunostained for immunofluorescence analysis. Note the lack of detection of dsRNA and nsp8 upon K22 treatment (4 µM) of cells infected with WT but not recombinant viruses. Scale bar is 10 µM.</p