Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was found initially in Wuhan, China in early December 2019. The pandemic has spread to 216 countries and regions, infecting more than 23310 000 people and causing over 800 000 deaths globally by Aug. 24, 2020, according to World Health Organization (https://www.who.int/emergencies/diseases/ novel-coronavirus-2019). Fever, cough, and dyspnea are the three common symptoms of the condition, whereas the conventional transmission route for SARS-CoV-2 is through droplets entering the respiratory tract. To date, infection control measures for COVID-19 have been focusing on the involvement of the respiratory system. However, ignoring potential faecal transmission and the gastrointestinal involvement of SARS-CoV-2 may result in mistakes in attempts to control the pandemic.
Betacoronavirus/isolation & purification* ; COVID-19 ; China/epidemiology* ; Coronavirus Infections/virology* ; Environmental Microbiology ; Feces/virology* ; Gastrointestinal Diseases/virology* ; Humans ; Models, Biological ; Pandemics ; Pneumonia, Viral/virology* ; RNA, Viral/genetics* ; SARS-CoV-2 ; Virus Shedding

Humans ; SARS Virus ; classification ; genetics ; pathogenicity ; Severe Acute Respiratory Syndrome ; virology

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
Antibodies, Viral ; immunology ; Bacteriophages ; genetics ; metabolism ; Epitopes ; genetics ; immunology ; Humans ; Membrane Glycoproteins ; genetics ; immunology ; Peptide Library ; SARS Virus ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; immunology

Animals ; Chiroptera ; virology ; Coronavirus ; classification ; genetics ; isolation & purification ; Coronavirus Infections ; transmission ; virology ; Databases, Genetic ; Genome, Viral ; Humans ; Male ; Middle Aged ; Middle East ; Phylogeny ; SARS Virus ; classification ; genetics ; isolation & purification ; Severe Acute Respiratory Syndrome ; virology

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.
Antibodies, Viral/*immunology ; Blotting, Western ; Coronavirus 229E, Human/*immunology ; Coronavirus OC43, Human/*immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins/genetics/*immunology ; Recombinant Proteins/immunology ; SARS Virus/genetics/*immunology ; Severe Acute Respiratory Syndrome/diagnosis/*immunology

Animals ; Humans ; RNA Interference ; RNA, Antisense ; genetics ; RNA, Small Interfering ; genetics ; SARS Virus ; genetics ; physiology ; Severe Acute Respiratory Syndrome ; virology

The main protease (M(pro)) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for trans-cleavage activity. However, the auto-processing mechanism of M(pro), i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of "immature" M(pro). Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature M(pro), are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the "immature" C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of M(pro) appears to require only two "immature" monomers approaching one another to form an "intermediate" dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of M(pro) from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M(pro).
Chromatography ; Circular Dichroism ; Cysteine Endopeptidases ; chemistry ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Polyproteins ; chemistry ; genetics ; metabolism ; Protein Multimerization ; SARS Virus ; chemistry ; enzymology ; genetics ; Spectrometry, Fluorescence ; Viral Proteins ; chemistry ; genetics ; metabolism

During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This "down-up-down" regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.
Amino Acid Sequence ; Crystallography, X-Ray ; DNA Primase ; chemistry ; genetics ; physiology ; Humans ; Isoenzymes ; chemistry ; genetics ; physiology ; Molecular Sequence Data ; Protein Structure, Secondary ; RNA, Viral ; biosynthesis ; SARS Virus ; chemistry ; genetics ; physiology ; Sequence Alignment ; Severe Acute Respiratory Syndrome ; virology ; Virus Replication

OBJECTIVETo construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.

METHODSS2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.

RESULTSThe pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.

CONCLUSIONSThe expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; Membrane Fusion ; drug effects ; Membrane Fusion Proteins ; biosynthesis ; isolation & purification ; Membrane Glycoproteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; SARS Virus ; genetics ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; biosynthesis ; genetics