A unique coronavirus severe acute respiratory syndrome-coronavirus (SARS-CoV) was revealed to be a causative agent of a lifethreatening SARS. Although this virus grows in a variety of tissues that express its receptor, the mechanism of the severe respiratory illness caused by this virus is not well understood. Here, we report a possible mechanism for the extensive damage seen in the major target organs for this disease. A recent study of the cell entry mechanism of SARS-CoV reveals that it takes an endosomal pathway. We found that proteases such as trypsin and thermolysin enabled SARS-CoV adsorbed onto the cell surface to enter cells directly from that site. This finding shows that SARS-CoV has the potential to take two distinct pathways for cell entry, depending on the presence of proteases in the environment. Moreover, the protease-mediated entry facilitated a 100-to 1,000-fold higher efficient infection than did the endosomal pathway used in the absence of proteases. These results suggest that the proteases produced in the lungs by inflammatory cells are responsible for high multiplication of SARS-CoV, which results in severe lung tissue damage. Likewise, elastase, a major protease produced in the lungs during inflammation, also enhanced SARS-CoV infection in cultured cells.cell entry ͉ protease ͉ spike protein ͉ SARS S evere acute respiratory syndrome (SARS) is caused by a SARS-associated coronavirus (SARS-CoV), a newly emergent member in a family of Coronaviridae (1-6). Unlike other human coronaviruses, SARS-CoV causes a fatal respiratory disease in humans (1-6). Coronavirus is an enveloped virus with a positive-stranded large genomic RNA with Ϸ30 kb (7). Spikes exist on the virion surface and resemble solar corona, each of which is composed of a trimer of the spike (S) protein (7,8). The S protein is a type I fusion protein of an approximate molecular weight of 180 kDa. The prototypical coronavirus mouse hepatitis virus enters into cells via the cell surface, although a variant isolated from persistent infection enters from an endosome, the low pH of which induces its fusion activity (9). However, the entry pathway of SARS-CoV appears to be distinct from that of the other coronaviruses. Simmons et al. (10) hypothesized that SARS-CoV enters cells by an endosomal pathway, and S protein is activated for fusion by trypsin-like protease in an acidic environment. This idea is based on the following two findings: (i) SARS-CoV infection can be blocked by lysosomotropic agents, and (ii) S protein expressed on cells is activated for fusion by trypsin. These results were obtained by studies using pseudotype retroviruses harboring SARS-CoV S protein on the envelope and those using S protein expressed on cells by expression vectors (10).In the present study, we show that various proteases, as well as trypsin, are effective in inducing the fusion of SARS-CoVinfected VeroE6 cells. These proteases facilitated SARS-CoV entry from the cell surface, which indicates that SARS-CoV has the potential to enter cells via two diff...

Steroid compounds, which are expected to have dual functions in blocking host inflammation and MERS-CoV replication, were screened from a chemical library. Within this library, ciclesonide, an inhaled corticosteroid, suppressed human coronavirus replication in cultured cells, but did not suppress replication of respiratory syncytial virus or influenza virus.The effective concentration of ciclesonide to block SARS-CoV-2 (the cause of replication (EC 90 ) was 6.3 μ M. After the eleventh consecutive MERS-CoV passage in the presence of ciclesonide, a resistant mutation was generated, which resulted in an amino acid substitution (A25V) in nonstructural protein (NSP) 15, as identified using reverse genetics. A recombinant virus with the mutation was also resistant to ciclesonide suppression of viral replication. These observations suggest that the effect of ciclesonide was specific to coronavirus, suggesting this is a candidate drug for treatment of patients suffering MERS or COVID-19.

Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and MERS-CoV replication. Ciclesonide, an inhaled corticosteroid, suppressed replication of MERS-CoV and other coronaviruses, including SARS-CoV-2, the cause of COVID-19, in cultured cells. The effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 μM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in non-structural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under the microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients. IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses replication of coronaviruses, including beta-coronaviruses (MHV-2, MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alpha-coronavirus (HCoV-229E), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for treatment of MERS and COVID-19.

The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions.

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