Human Coronavirus HCoV-229E Research Articles

Antiviral miliusanes and isolation of an unprecedented miliusane dimer from Miliusa balansae

In an extensive screening endeavor for anti-coronaviral compounds, we examined 824 tropical plant extracts from the Annonaceae and Rutaceae families. The screening identified an ethyl acetate extract from the aerial parts of Miliusa balansae for its potent inhibitory activity against Human coronavirus HCoV-229E. Subsequent bioassay-guided fractionation of this extract revealed two unreported miliusanes including a complex dimeric structure and seven known compounds, comprising miliusane XXXVI, (+)-miliusol, bistyryls, styryl-pyranones, and the flavonoid rhamnetin. The absolute configuration of the new dimeric miliusane was determined by X-ray crystallography and a putative biogenetic origin was proposed. Investigation of the antiviral effect of these nine phytochemicals within HCoV-229E-infected Huh-7 cells showed that (+)-miliusol and miliusane XXXVI exert antiviral activity at non-cytotoxic concentrations, with IC50 values of 1.15 μM and 19.20 μM, respectively. Furthermore, these compounds significantly inhibited SARS-CoV-2 infection in Vero cells, presenting IC50 values of 11.31 μM for (+)-miliusol and 17.92 μM for miliusane XXXVI. Additionally, both compounds exhibited a potent antiviral effect against the emergent mosquito-borne Zika virus, with IC50 values of 1.34 μM and 23.45 μM, respectively. Time-of-addition assays suggest that their mechanism of action might target later stages of the viral cycle, indicating potential modulation of specific cellular pathways. These findings reinforce the invaluable contribution of medicinal flora as reservoirs of natural antiviral agents and emphasize their prospective role in combatting viruses of medical interest.

Lichen or Associated Micro-Organism Compounds Are Active against Human Coronaviruses.

(1) Background: Since the emergence of SARS-CoV-2, responsible for the COVID-19 pandemic, efforts have been made to identify antiviral compounds against human coronaviruses. With the aim of increasing the diversity of molecule scaffolds, 42 natural compounds, of which 28 were isolated from lichens and 14 from their associated microorganisms (bacteria and fungi), were screened against human coronavirus HCoV-229E. (2) Methods: Antiviral assays were performed using HCoV-229E in Huh-7 and Huh-7/TMPRSS2 cells and SARS-CoV-2 in a Vero-81-derived clone with a GFP reporter probe. (3) Results: Four lichen compounds, including chloroatranol, emodin, perlatolic acid and vulpinic acid, displayed high activities against HCoV-229E (IC50 = 68.86, 59.25, 16.42 and 14.58 μM, respectively) and no toxicity at active concentrations. Kinetics studies were performed to determine their mode of action. The four compounds were active when added at the replication step. Due to their significant activity, they were further tested on SARS-CoV-2. Perlatolic acid was shown to be active against SARS-CoV-2. (4) Conclusions: Taken together, these results show that lichens are a source of interesting antiviral agents against human coronaviruses. Moreover, perlatolic acid might be further studied for its pan-coronavirus antiviral activity.

A Comparative Study of the Antiviral Properties of Thermally Sprayed Coatings against Human Coronavirus HCoV-229E

For decades, novel viral strains of respiratory tract infections have caused human pandemics and initiated widespread illnesses. The recent coronavirus disease 2019 (COVID-19) outbreak caused by the SARS-CoV-2 virus has raised an urgent need to develop novel antiviral coatings as one of the potential solutions to mitigate the transmission of viral pathogens. Titanium dioxide is considered an excellent candidate for viral disinfection under light irradiation, with the potential to be activated under visible light for indoor applications. This research assessed the antiviral performance of thermally sprayed TiO2 coatings under UVA and ambient light. We also report the antiviral performance of TiO2 composites with other oxides, such as Cu2O and Al2O3, produced by suspension plasma spray, atmospheric plasma spray, and suspension high-velocity oxygen fuel techniques. To evaluate the antiviral performance of the above coatings in a containment level-2 laboratory, a human common cold coronavirus, HCoV-229E, was initially used as a relevant surrogate for SARS-CoV-2. Coatings were also analyzed using SEM and XRD and were classified based on their surface roughness, porosity, and phase composition. Collectively, the thermally sprayed coatings showed comparable or slightly better antiviral activity compared to copper. The most significant level of activity observed was approximately 20% to 50% higher than that of a pure copper plate.

Luteolin Isolated from Juncus acutus L., a Potential Remedy for Human Coronavirus 229E.

The COVID-19 pandemic, caused by SARS-CoV-2, addressed the lack of specific antiviral drugs against coronaviruses. In this study, bioguided fractionation performed on both ethyl acetate and aqueous sub-extracts of Juncus acutus stems led to identifying luteolin as a highly active antiviral molecule against human coronavirus HCoV-229E. The apolar sub-extract (CH2Cl2) containing phenanthrene derivatives did not show antiviral activity against this coronavirus. Infection tests on Huh-7 cells, expressing or not the cellular protease TMPRSS2, using luciferase reporter virus HCoV-229E-Luc showed that luteolin exhibited a dose-dependent inhibition of infection. Respective IC50 values of 1.77 µM and 1.95 µM were determined. Under its glycosylated form (luteolin-7-O-glucoside), luteolin was inactive against HCoV-229E. Time of addition assay showed that utmost anti-HCoV-229E activity of luteolin was achieved when added at the post-inoculation step, indicating that luteolin acts as an inhibitor of the replication step of HCoV-229E. Unfortunately, no obvious antiviral activity for luteolin was found against SARS-CoV-2 and MERS-CoV in this study. In conclusion, luteolin isolated from Juncus acutus is a new inhibitor of alphacoronavirus HCoV-229E.

Clear polyurethane coatings with excellent virucidal properties: Preparation, characterization and rapid inactivation of human coronaviruses 229E and SARS-CoV-2

Commercial polyurethane (PU) coating formulations have been modified with 1-(hydroxymethyl)-5,5-dimethylhydantoin (HMD) both in bulk (0.5 and 1% w/w) and onto the coatings surface as an N-halamine precursor, to obtain clear coatings with high virucidal activity. Upon immersion in diluted chlorine bleaching, the hydantoin structure on the grafted PU membranes was transformed into N-halamine groups, with a high surface chlorine concentration (40-43μg/cm2). Fourier transform infrared spectroscopy (FTIR) spectroscopy, thermogravimetric analysis (TGA), energy-dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS) and iodometric titration were used to characterize the coatings and quantify the chlorine contents of the PU membranes after chlorination. Biological evaluation of their activity against Staphylococcus aureus (Gram-positive bacteria) and human coronaviruses HCoV-229E and SARS-CoV-2 was performed, and high inactivation of these pathogens was observed after short contact times. The inactivation of HCoV-229E was higher than 98% for all modified samples after just 30 minutes, whereas it was necessary 12 hours of contact time for complete inactivation of SARS-CoV-2. The coatings were fully rechargeable by immersion in diluted chlorine bleach (2% v/v) for at least 5 chlorination-dechlorination cycles. Moreover, the performance of the antivirus efficiency of the coatings is considered as long-lasting, because experiments of reinfection of the coatings with HCoV-229E coronavirus did not show any loss of the virucidal activity after three consecutive infection cycles without reactivation of the N-halamine groups.

Antiviral functionalization of a polypropylene nonwoven textile structure as a self-decontaminating layer for respiratory masks.

The aim of this work was to develop a filtering biocidal polypropylene (PP) nonwoven textile structure to block and inactivate airborne bacteria and viruses. PP filters were functionalized with a cyclodextrin (CD)-polycarboxylic acid-crosslinked polymer (PP-CD) through a pad/dry/curing process, and were then activated by padding in an alkyl dimethyl benzalkonium chloride (ADBAC) solution. The textile finishing process parameters were optimized with the perspective of mass production, considering the threshold temperature necessary for provoking crosslinking and the limitation of the low thermal stability of PP. The use of an aqueous solution containing hydroxypropyl-β-cyclodextrin (HPβCD), 1,2,3,4-butanetetracarboxylic acid (BTCA), ammonium hypophosphite (AH), and a surfactant allowed immobilization of the optimal quantity of cyclodextrin polymer under curing for 5 minutes at 125 °C without affecting the nonwoven PP structure. The presence of CD drastically increased the sorption of ADBAC on the textiles. There was leaching of ADBAC at the first rinsing and then satisfactory fastness at the second and third rinsings, revealing adsorption mechanisms by weak physical interactions, ionic interactions, and inclusion of ADBAC inside the CD cavities. SEM revealed no clogging of the nonwoven pores, nor any increase in the air flow resistance, as evaluated by pressure drop measurements. The filtration efficiency of particulate matter PM3.0 and PM0.5 was moderately affected, in contrast to that of PM0.3, which greatly decreased due to the loss of the electrostatic charge of the filter upon the functionalization process. Bactericidal tests resulted in a reduction of 3 log10 against Staphylococcus aureus, and for virucidal tests on human coronavirus HCoV-229E, there was a reduction of 3.4 log10, with both strains undergoing 20 minutes of contact. Finally, the filter we developed is manufacturable by a scalable process, and because of its filtration and biocidal performances, it is a choice material as a self-disinfecting layer in the fabrication of facepiece respirators.

Discovery of 2-Phenylquinolines with Broad-Spectrum Anti-coronavirus Activity.

A selection of compounds from a proprietary library, based on chemical diversity and various biological activities, was evaluated as potential inhibitors of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a phenotypic-based screening assay. A compound based on a 2-phenylquinoline scaffold emerged as the most promising hit, with EC50 and CC50 values of 6 and 18 μM, respectively. The subsequent selection of additional analogues, along with the synthesis of ad hoc derivatives, led to compounds that maintained low μM activity as inhibitors of SARS-CoV-2 replication and lacked cytotoxicity at 100 μM. In addition, the most promising congeners also show pronounced antiviral activity against the human coronaviruses HCoV-229E and HCoV-OC43, with EC50 values ranging from 0.2 to 9.4 μM. The presence of a 6,7-dimethoxytetrahydroisoquinoline group at the C-4 position of the 2-phenylquinoline core gave compound 6g that showed potent activity against SARS-CoV-2 helicase (nsp13), a highly conserved enzyme, highlighting a potentiality against emerging HCoVs outbreaks.

Integrated hepatic single-cell RNA sequencing and untargeted metabolomics reveals the immune and metabolic modulation of Qing-Fei-Pai-Du decoction in mice with coronavirus-induced pneumonia

BackgroundAlthough Qing-Fei-Pai-Du decoction (QFPDD) is extensively used clinically to treat COVID-19 patients, the mechanism by which it modulates the immunological and metabolic functions of liver tissue remains unknown. PurposeThe purpose of this study is to investigate the mechanism of action of QFPDD in the treatment of mice with coronavirus-induced pneumonia by combining integrated hepatic single-cell RNA sequencing and untargeted metabolomics. MethodsWe developed a human coronavirus pneumonia model in BALB/c mice by infecting them with human coronavirus HCoV-229E with stimulating them with cold-damp environment. We initially assessed the status of inflammation and immunity in model mice treated with or without QFPDD by detecting peripheral blood lymphocytes and inflammatory cytokines. Then, single-cell RNA sequencing and untargeted metabolomics were performed on mouse liver tissue. ResultsHCoV-229E infection in combination with exposure to a cold-damp environment significantly decreased the percentage of peripheral blood lymphocytes (CD4+ and CD8+ T cells, B cells) in mice, which was enhanced by QFPDD therapy. Meanwhile, the levels of inflammatory cytokines such as IL-6, TNF-α, and IFN-γ were significantly increased in mouse models but significantly decreased by QFPDD treatment. Single-cell RNA sequencing analysis showed that QFPDD could attenuate disease-associated alterations in gene expression, core transcriptional regulatory networks, and cell-type composition. Computational predictions indicated that QFPDD rectified the observed aberrant patterns of cell-cell communication. Additionally, the metabolic profiles of liver tissue in the Model group were distinct from mice in the Control group, and QFPDD significantly regulated hepatic purine metabolism. ConclusionTo the best of our knowledge, this study is the first to integrate hepatic single-cell RNA sequencing and untargeted metabolomics into a TCM formula and these valuable findings indicate that QFPDD can improve immune function and reduce liver injury and inflammation.

SPR-Based Kinetic Analysis of the Early Stages of Infection in Cells Infected with Human Coronavirus and Treated with Hydroxychloroquine.

Cell-based assays are a valuable tool for examination of virus–host cell interactions and drug discovery processes, allowing for a more physiological setting compared to biochemical assays. Despite the fact that cell-based SPR assays are label-free and thus provide all the associated benefits, they have never been used to study viral growth kinetics and to predict drug antiviral response in cells. In this study, we prove the concept that the cell-based SPR assay can be applied in the kinetic analysis of the early stages of viral infection of cells and the antiviral drug activity in the infected cells. For this purpose, cells immobilized on the SPR slides were infected with human coronavirus HCov-229E and treated with hydroxychloroquine. The SPR response was measured at different time intervals within the early stages of infection. Methyl Thiazolyl Tetrazolium (MTT) assay was used to provide the reference data. We found that the results of the SPR and MTT assays were consistent, and SPR is a reliable tool in investigating virus–host cell interaction and the mechanism of action of viral inhibitors. SPR assay was more sensitive and accurate in the first hours of infection within the first replication cycle, whereas the MTT assay was not so effective. After the second replication cycle, noise was generated by the destruction of the cell layer and by the remnants of dead cells, and masks useful SPR signals.

Assessment of Antiviral Coatings for High-Touch Surfaces by Using Human Coronaviruses HCoV-229E and SARS-CoV-2.

ABSTRACTA novel and robust approach to evaluate the antiviral activity of coatings was developed, assessing three commercially available leave-on surface coating products for efficacy against human coronaviruses (HCoVs) HCoV-229E and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The assessment is based on three criteria that reflect real-life settings, namely, (i) immediate antiviral effect, (ii) effect after repeated cleaning of the coated surface, and (iii) antiviral activity in the presence of organic material. The results showed that only a copper compound-based coating successfully met all three criteria. A quaternary ammonium compound-based coating did not meet the second criterion, and a coating based on reactive oxygen species showed no antiviral effect. Moreover, the study demonstrated that HCoV-229E is a relevant SARS-CoV-2 surrogate for such experiments. This new approach allows benchmarking of currently available antiviral coatings and future coating developments to avoid unjustified claims. The deployment of efficient antiviral coatings can offer an additional measure to mitigate the risk of transmission of respiratory viruses like SARS-CoV-2 or influenza viruses from high-touch surfaces.IMPORTANCE SARS-CoV-2, the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, is transmitted mainly person-to-person through respiratory droplets, while the contribution of fomite transmission is less important than suspected at the beginning of the pandemic. Nevertheless, antiviral-coating solutions can offer an additional measure to mitigate the risk of SARS-CoV-2 transmission from high-touch surfaces. The deployment of antiviral coatings is not new, but what is currently lacking is solid scientific evidence of the efficacy of commercially available self-disinfecting surfaces under real-life conditions. Therefore, we developed a novel, robust approach to evaluate the antiviral activity of such coatings, applying strict quality criteria to three commercially available products to test their efficacies against SARS-CoV-2. We also showed that HCoV-229E is a relevant surrogate for such experiments. Our approach will also bring significant benefit to the evaluation of the effects of coatings on the survival of nonenveloped viruses, which are known to be more tolerant to desiccation and disinfectants and for which high-touch surfaces play an important role.

Multi-Omics-Guided Discovery of Omicsynins Produced by Streptomyces sp. 1647: Pseudo-Tetrapeptides Active Against Influenza A Viruses and Coronavirus HCoV-229E

Many microorganisms have mechanisms that protect cells against attack from viruses. The fermentation components of Streptomyces sp. 1647 exhibit potent anti-influenza A virus (IAV) activity. This strain was isolated from soil in southern China in the 1970s, but the chemical nature of its antiviral substance(s) has remained unknown until now. We used an integrated multi-omics strategy to identify the antiviral agents from this streptomycete. The antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) analysis of its genome sequence revealed 38 biosynthetic gene clusters (BGCs) for secondary metabolites, and the target BGCs possibly responsible for the production of antiviral components were narrowed down to three BGCs by bioactivity-guided comparative transcriptomics analysis. Through bioinformatics analysis and genetic manipulation of the regulators and a biosynthetic gene, cluster 36 was identified as the BGC responsible for the biosynthesis of the antiviral compounds. Bioactivity-based molecular networking analysis of mass spectrometric data from different recombinant strains illustrated that the antiviral compounds were a class of structural analogues. Finally, 18 pseudo-tetrapeptides with an internal ureido linkage, omicsynins A1–A6, B1–B6, and C1–C6, were identified and/or isolated from fermentation broth. Among them, 11 compounds (omicsynins A1, A2, A6, B1–B3, B5, B6, C1, C2, and C6) are new compounds. Omicsynins B1–B4 exhibited potent antiviral activity against IAV with the 50% inhibitory concentration (IC50) of approximately 1 µmol∙L–1 and a selectivity index (SI) ranging from 100 to 300. Omicsynins B1–B4 also showed significant antiviral activity against human coronavirus HCoV-229E. By integrating multi-omics data, we discovered a number of novel antiviral pseudo-tetrapeptides produced by Streptomyces sp. 1647, indicating that the secondary metabolites of microorganisms are a valuable source of novel antivirals.

Evaluation of Antiviral, Antibacterial and Antiproliferative Activities of the Endophytic Fungus Curvularia papendorfii, and Isolation of a New Polyhydroxyacid.

An endophytic fungus isolated from Vernonia amygdalina, a medicinal plant from Sudan, was taxonomically characterized as Curvularia papendorfii. Ethyl acetate crude extract of C. papendorfii revealed an important antiviral effect against two viral pathogens, the human coronavirus HCoV 229E and a norovirus surrogate, the feline coronavirus FCV F9. For the last one, 40% of the reduction of the virus-induced cytopathogenic effect at lower multiplicity of infection (MOI) 0.0001 was observed. Selective antibacterial activity was obtained against Staphylococcus sp. (312 µg/mL), and interesting antiproliferative activity with half maximal inhibitory concentration (IC50) value of 21.5 ± 5.9 µg/mL was observed against human breast carcinoma MCF7 cell line. Therefore, C. papendorfii crude extract was further investigated and fractionated. Twenty-two metabolites were identified by gas chromatography coupled to mass spectrometry (GC–MS), and two pure compounds, mannitol and a new polyhydroxyacid, called kheiric acid, were characterized. A combination of spectroscopic methods was used to elucidate the structure of the new aliphatic carboxylic acid: kheiric acid (3,7,11,15-tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl-hexacosa-4E,8E,12E,16,18-pentaenoic acid). Kheiric acid showed an interesting result with a minimum inhibitory concentration (MIC) value of 62.5 µg/mL against meticillin-resistant Staphylococcus aureus (MRSA). Hence, endophytes associated with medicinal plants from Sudan merit more attention, as they could be a treasure of new bioactive compounds.

The expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus.

BackgroundIn the absence of effective drugs, controlling SARS relies on the rapid identification of cases and appropriate management of the close contacts, or effective vaccines for SARS. Therefore, developing specific and sensitive laboratory tests for SARS as well as effective vaccines are necessary for national authorities.ResultsGenes encoding truncated nucleocapsid (N) and spike (S) proteins of SARSCoV were cloned into the expression vector pQE30 and fusionally expressed in Escherichia coli M15. The fusion protein was analyzed for reactivity with SARS patients' sera and with anti-sera against the two human coronaviruses HCoV 229E and HCoV OC43 by ELISA, IFA and immunoblot assays. Furthermore, to evaluate the antigen-specific humoral antibody and T-cell responses in mice, the fusion protein was injected into 6-week-old BALB/c mice and a neutralization test as well as a T-cell analysis was performed. To evaluate the antiviral efficacy of immunization, BALB/c mice were challenged intranasally with SARSCoV at day 33 post injection and viral loads were determined by fluorescent quantitative RT-PCR. Serological results showed that the diagnostic sensitivity and specificity of the truncated S-N fusion protein derived the SARS virus were > 99% (457/460) and 100.00% (650/650), respectively. Furthermore there was no cross-reactivity with other two human coronaviruses. High titers of antibodies to SRASCoV appeared in the immunized mice and the neutralization test showed that antibodies to the fusion protein could inhibit SARSCoV. The T cell proliferation showed that the fusion protein could induce an antigen-specific T-cell response. Fluorescent quantitative RT-PCR showed that BALB/c mice challenged intranasally with SARSCoV at day 33 post injection were completely protected from virus replication.ConclusionThe truncated S-N fusion protein is a suitable immunodiagnostic antigen and vaccine candidate.

Antiviral Strategies Against Human Coronaviruses

Since the mid 60's the human coronaviruses (HCoV), represented by HCoV-OC43 and HCoV-229E, were generally considered relatively harmless viruses. This status changed dramatically with the emergence of SARS-CoV in 2002/2003. The SARS-CoV pandemic took 774 lives around the globe and infected more than 8000 people in 29 countries. SARS-CoV is believed to be of zoonotic origin, transmitted from its natural reservoir in bats through several animal species (e.g., civet cats, raccoon dogs sold for human consumption in markets in southern China). The epidemic was halted in 2003 by a highly effective global public health response, and SARS-CoV is currently not circulating in humans. The outbreak of SARS-CoV and the danger of its re-introduction into the human population, as well as the danger of the emergence of other zoonotic coronaviral infections triggered an intense survey for an efficient treatment that resulted in the evaluation of several anticoronaviral compounds. HCoV-NL63 and HCoV-HKU1 were identified shortly after the SARS-CoV outbreak. The 4 human coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 cause mild respiratory illnesses when compared to SARS, but these infections are involved in 10 - 20 % of hospitalizations of young children and immunocompromised adults with respiratory tract illness. Therefore, there is an urgent need for a successful therapy to prevent disease induction or a vaccine to prevent new infections. This review summarizes the current status of anticoronaviral strategies.

Mutational Analysis of Aminopeptidase N, a Receptor for Several Group 1 Coronaviruses, Identifies Key Determinants of Viral Host Range

Feline coronavirus (FCoV), porcine transmissible gastroenteritis coronavirus (TGEV), canine coronavirus (CCoV), and human coronavirus HCoV-229E, which belong to the group 1 coronavirus, use aminopeptidase N (APN) of their natural host and feline APN (fAPN) as receptors. Using mouse-feline APN chimeras, we identified three small, discontinuous regions, amino acids (aa) 288 to 290, aa 732 to 746 (called R1), and aa 764 to 788 (called R2) in fAPN that determined the host ranges of these coronaviruses. Blockade of infection with anti-fAPN monoclonal antibody RG4 suggested that these three regions lie close together on the fAPN surface. Different residues in fAPN were required for infection with each coronavirus. HCoV-229E infection was blocked by an N-glycosylation sequon present between aa 288 to 290 in murine APN. TGEV required R1 of fAPN, while FCoV and CCoV required both R1 and R2 for entry. N740 and T742 in fAPN and the homologous R741 in human APN (hAPN) were key determinants of host range for FCoV, TGEV, and CCoV. Residue N740 in fAPN was essential only for CCoV receptor activity. A conservative T742V substitution or a T742R substitution in fAPN destroyed receptor activity for the pig, dog, and cat coronaviruses, while a T742S substitution retained these receptor activities. Thus, the hydroxyl on T742 is required for the coronavirus receptor activity of fAPN. In hAPN an R741T substitution caused a gain of receptor activity for TGEV but not for FCoV or CCoV. Therefore, entry and host range of these group 1 coronaviruses depend on the ability of the viral spike glycoproteins to recognize small, species-specific amino acid differences in the APN proteins of different species.

Homology Models and Molecular Dynamics Simulations of Main Proteinase from Coronavirus Associated with Severe Acute Respiratory Syndrome (SARS).

In this study, two structural models (denoted as MproST and MproSH) of the main proteinase (Mpro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS‐CoV) were constructed based on the crystallographic structures of Mpro from transmissible gastroenteritis coronavirus (TGEV) (MproT) and human coronavirus HcoV‐229E (MproH), respectively. Various 200 ps molecular dynamics simulations were subsequently performed to investigate the dynamics behaviors of several structural features. Both MproST and MproSH exhibit similar folds as their respective template proteins. These structural models reveal three distinct functional domains as well as an intervening loop connecting domains II and III as found in both template proteins. In addition, domain III of these structures exhibits the least secondary structural conservation. A catalytic cleft containing the substrate binding subsites S1 and the S2 between domains I and II are also observed in these structural models. Although these structures share many common features, the most significant difference occurs at the S2 subsite, where the amino acid residues lining up this subsite are least conserved. It may be a critical challenge for designing anti‐SARS drugs by simply screening the known database of proteinase inhibitors.

Molecular Dynamics Simulations of Various Coronavirus Main Proteinases

In this study, two homology models (denoted as MproST and MproSH) of main proteinase (Mpro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed based on the crystal structures of Mpro from transmissible gastroenteritis coronavirus (TGEV) (MproT) and human coronavirus HcoV-229E (MproH), respectively. Both MproST and MproSH exhibit similar folds as their respective template proteins. These homology models reveal three distinct functional domains as well as an intervening loop connecting domains II and III as found in both template proteins. A catalytic cleft containing the substrate binding sites S1 and S2 between domains I and II are also observed. S2 undergoes more significant structural fluctuation than S1 during the 400 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft, while S1 is situated in the very bottom of this cleft. The thermal unfolding of these proteins begins at domain III, where the structure is least conserved among these proteins. Mpro may still maintain its proteolytic activity while it is partially unfolded. The electrostatic interaction between Arg40 and Asp186 plays an important role in maintaining the structural integrity of both S1 and S2.