Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

Sporadic Occurrence of Ensitrelvir-Resistant SARS-CoV-2, Japan.

Using the GISAID EpiCoV database, we identified 256 COVID-19 patients in Japan during March 31-December 31, 2023, who had mutations in the SARS-CoV-2 nonstructural protein 5 conferring ensitrelvir resistance. Ongoing genomic surveillance is required to monitor emergence of SARS-CoV-2 mutations that are resistant to anticoronaviral drugs.

The anti-influenza virus drug favipiravir has little effect on replication of SARS-CoV-2 in cultured cells.

Favipiravir (T-705, commercial name Avigan), a drug developed to treat influenza virus infection, has been used in some countries as an oral treatment for COVID-19; however, its clinical efficacy in this context is controversial.….

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.

The Inhaled Steroid Ciclesonide Blocks SARS-CoV-2 RNA Replication by Targeting the Viral Replication-Transcription Complex in Cultured Cells.

Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), in cultured cells. The 90% 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 nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a 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 the replication of coronaviruses, including betacoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphacoronavirus (human coronavirus 229E [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 the treatment of MERS and COVID-19.

Environmental Sampling for Severe Acute Respiratory Syndrome Coronavirus 2 During a COVID-19 Outbreak on the Diamond Princess Cruise Ship.

During a COVID-19 outbreak on the Diamond Princess cruise ship we sampled environmental surfaces after passengers and crew vacated cabins. SARS-CoV-2 RNA was detected in 58 of 601 samples (10%) from case cabins 1-17 days after cabins were vacated but not from noncase cabins. There was no difference in detection proportion between cabins of symptomatic (15%, 28/189; cycle quantification [Cq], 29.79-38.86) and asymptomatic cases (21%, 28/131; Cq, 26.21-38.99). No SARS-CoV-2 virus was isolated from any of the samples. Transmission risk of SARS-CoV-2 from symptomatic and asymptomatic patients may be similar and surfaces could be involved in transmission.

Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion.

A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses.IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

Antiviral activities of mycophenolic acid and IMD-0354 against SARS-CoV-2.

In this study, the anti-severe acute respiratory syndrome coronavirus-2 (anti-SARS-CoV-2) activity of mycophenolic acid (MPA) and IMD-0354 was analyzed. These compounds were chosen based on their antiviral activities against other coronaviruses. Because they also inhibit dengue virus (DENV) infection, other anti-DENV compounds/drugs were also assessed. On SARS-CoV-2-infected VeroE6/TMPRSS2 monolayers, both MPA and IMD-0354, but not other anti-DENV compounds/drugs, showed significant anti-SARS-CoV-2 activity. Although MPA reduced the viral RNA level by only approximately 100-fold, its half maximal effective concentration was as low as 0.87 µ m, which is easily achievable at therapeutic doses of mycophenolate mofetil. MPA targets the coronaviral papain-like protease and an in-depth study on its mechanism of action would be useful in the development of novel anti-SARS-CoV-2 drugs.

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry.

Human coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence or absence of protease inhibitors. We found that, compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased so that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin. IMPORTANCE: Many enveloped viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease TMPRSS2 rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for coronavirus-mediated diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), should target cell surface TMPRSS2 rather than endosomal cathepsin.

Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay.

Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhea virus, but promotes its infectivity via aminopeptidase activity.

Porcine epidemic diarrhea virus (PEDV), a causative agent of pig diarrhoea, has recently caused significant economic damage worldwide. Porcine aminopeptidase N (pAPN) has been reported to be the receptor for PEDV, although robust evidence is lacking. In the present study, we explored whether pAPN functions as a receptor for PEDV. Human HeLa cells expressing pAPN and pAPN-positive porcine CPK cells failed to support PEDV infection, but were susceptible to infection by transmissible gastroenteritis virus (TGEV), which utilizes pAPN as a functional receptor. In contrast to TGEV, PEDV did not bind soluble porcine aminopeptidases (pAPs) and infection was not inhibited by the soluble form of pAPs. However, overexpression of pAPN in porcine CPK cells (CPK-pAPN cells) slightly increased the production of PEDV, and the increased replication in CPK-pAPN cells was inhibited by bestatin, an inhibitor of the protease activity of aminopeptidase N. These results suggest that pAPN is not a functional receptor for PEDV, but promotes the infection of PEDV through its protease activity.

Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2.

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.

TMPRSS2 is an activating protease for respiratory parainfluenza viruses.

Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.

Possible involvement of infection with human coronavirus 229E, but not NL63, in Kawasaki disease.

Although human coronavirus (HCoV)-NL63 was once considered a possible causative agent of Kawasaki disease based on RT-PCR analyses, subsequent studies could not confirm the result. In this study, this possibility was explored using serological tests. To evaluate the role of HCoV infection in patients with Kawasaki disease, immunofluorescence assays and virus neutralizing tests were performed. Paired serum samples were obtained from patients with Kawasaki disease who had not been treated with γ-globulin. HCoV-NL63 and two antigenically different isolates of HCoV-229E (ATCC-VR740 and a new isolate, Sendai-H) were examined as controls. Immunofluorescence assays detected no difference in HCoV-NL63 antibody positivity between the patients with Kawasaki disease and controls, whereas the rate of HCoV-229E antibody positivity was higher in the patients with Kawasaki disease than that in controls. The neutralizing tests revealed no difference in seropositivity between the acute and recovery phases of patients with Kawasaki disease for the two HCoV-229Es. However, the Kawasaki disease specimens obtained from patients in recovery phase displayed significantly higher positivity for Sendai-H, but not for ATCC-VR740, as compared to the controls. The serological test supported no involvement of HCoV-NL63 but suggested the possible involvement of HCoV-229E in the development of Kawasaki disease.

Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections. METHODS: Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region. RESULTS: The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR. CONCLUSIONS: These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.

Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry.

The type II transmembrane protease TMPRSS2 activates the spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) on the cell surface following receptor binding during viral entry into cells. In the absence of TMPRSS2, SARS-CoV achieves cell entry via an endosomal pathway in which cathepsin L may play an important role, i.e., the activation of spike protein fusogenicity. This study shows that a commercial serine protease inhibitor (camostat) partially blocked infection by SARS-CoV and human coronavirus NL63 (HCoV-NL63) in HeLa cells expressing the receptor angiotensin-converting enzyme 2 (ACE2) and TMPRSS2. Simultaneous treatment of the cells with camostat and EST [(23,25)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester], a cathepsin inhibitor, efficiently prevented both cell entry and the multistep growth of SARS-CoV in human Calu-3 airway epithelial cells. This efficient inhibition could be attributed to the dual blockade of entry from the cell surface and through the endosomal pathway. These observations suggest camostat as a candidate antiviral drug to prevent or depress TMPRSS2-dependent infection by SARS-CoV.