Sars-CoV-2 Envelope and Membrane Proteins: Structural Differences Linked to Virus Characteristics?

The Coronavirus Disease 2019 (COVID-19) is a new viral infection caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2). Genomic analyses have revealed that SARS-CoV-2 is related to Pangolin and Bat coronaviruses. In this report, a structural comparison between the Sars-CoV-2 Envelope and Membrane proteins from different human isolates with homologous proteins from closely related viruses is described. The analyses here reported show the high structural similarity of Envelope and Membrane proteins to the counterparts from Pangolin and Bat coronavirus isolates. However, the comparisons have also highlighted structural differences specific of Sars-CoV-2 proteins which may be correlated to the cross-species transmission and/or to the properties of the virus. Structural modelling has been applied to map the variant sites onto the predicted three-dimensional structure of the Envelope and Membrane proteins.

Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.

Discovery of a novel coronavirus, China Rattus coronavirus HKU24, from Norway rats supports the murine origin of Betacoronavirus 1 and has implications for the ancestor of Betacoronavirus lineage A.

UNLABELLED: We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (ßCoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1, and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B ßCoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by ßCoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1, and rodents are likely an important reservoir for ancestors of lineage A ßCoVs. IMPORTANCE: While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A ßCoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A ßCoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China with a high seroprevalence. The unique genome features and phylogenetic analysis supported the suggestion that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and being distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble those of both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1, with interspecies transmission from rodents to other mammals having occurred centuries ago, before the emergence of human coronavirus (HCoV) OC43 in the late 1800s. Rodents are likely an important reservoir for ancestors of lineage A ßCoVs.

A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein.

UNLABELLED: Prophylactic and therapeutic strategies are urgently needed to combat infections caused by the newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have developed a neutralizing monoclonal antibody (MAb), designated Mersmab1, which potently blocks MERS-CoV entry into human cells. Biochemical assays reveal that Mersmab1 specifically binds to the receptor-binding domain (RBD) of the MERS-CoV spike protein and thereby competitively blocks the binding of the RBD to its cellular receptor, dipeptidyl peptidase 4 (DPP4). Furthermore, alanine scanning of the RBD has identified several residues at the DPP4-binding surface that serve as neutralizing epitopes for Mersmab1. These results suggest that if humanized, Mersmab1 could potentially function as a therapeutic antibody for treating and preventing MERS-CoV infections. Additionally, Mersmab1 may facilitate studies of the conformation and antigenicity of MERS-CoV RBD and thus will guide rational design of MERS-CoV subunit vaccines. IMPORTANCE: MERS-CoV is spreading in the human population and causing severe respiratory diseases with over 40% fatality. No vaccine is currently available to prevent MERS-CoV infections. Here, we have produced a neutralizing monoclonal antibody with the capacity to effectively block MERS-CoV entry into permissive human cells. If humanized, this antibody may be used as a prophylactic and therapeutic agent against MERS-CoV infections. Specifically, when given to a person (e.g., a patient's family member or a health care worker) either before or after exposure to MERS-CoV, the humanized antibody may prevent or inhibit MERS-CoV infection, thereby stopping the spread of MERS-CoV in humans. This antibody can also serve as a useful tool to guide the design of effective MERS-CoV vaccines.

MERS-CoV papain-like protease has deISGylating and deubiquitinating activities.

Coronaviruses encode papain-like proteases (PLpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (DUB)/deISGylating activity, which is hypothesized to modify the innate immune response to infection. Here, we investigate the predicted DUB activity of the PLpro domain of the recently described Middle East Respiratory Syndrome Coronavirus (MERS-CoV). We found that expression of MERS-CoV PLpro reduces the levels of ubiquitinated and ISGylated host cell proteins; consistent with multifunctional PLpro activity. Further, we compared the ability of MERS-CoV PLpro and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) PLpro to block innate immune signaling of proinflammatory cytokines. We show that expression of SARS-CoV and MERS-CoV PLpros blocks upregulation of cytokines CCL5, IFN-ß and CXCL10 in stimulated cells. Overall these results indicate that the PLpro domains of MERS-CoV and SARS-CoV have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis.

Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease.

The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-ß by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.

Functional analysis of the murine coronavirus genomic RNA packaging signal.

Coronaviruses selectively package genomic RNA into assembled virions, despite the great molar excess of subgenomic RNA species that is present in infected cells. The genomic packaging signal (PS) for the coronavirus mouse hepatitis virus (MHV) was originally identified as an element that conferred packaging capability to defective interfering RNAs. The MHV PS is an RNA structure that maps to the region of the replicase gene encoding the nonstructural protein 15 subunit of the viral replicase-transcriptase complex. To begin to understand the role and mechanism of action of the MHV PS in its native genomic locus, we constructed viral mutants in which this cis-acting element was altered, deleted, or transposed. Our results demonstrated that the PS is pivotal in the selection of viral genomic RNA for incorporation into virions. Mutants in which PS RNA secondary structure was disrupted or entirely ablated packaged large quantities of subgenomic RNAs, in addition to genomic RNA. Moreover, the PS retained its function when displaced to an ectopic site in the genome. Surprisingly, the PS was not essential for MHV viability, nor did its elimination have a severe effect on viral growth. However, the PS was found to provide a distinct selective advantage to MHV. Viruses containing the PS readily outcompeted their otherwise isogenic counterparts lacking the PS.

Isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14, from domestic rabbits.

We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 10(8) copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5'-UCUAAAC-3'. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1″-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that RbCoV HKU14 should represent a separate species. RbCoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (RdRp) genes dated the most recent common ancestor of RbCoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against RbCoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

Characterization of the stunting syndrome agent: physicochemical properties.

Stunting syndrome is an enteric disease of turkeys causing diarrhea, reduced weight gain, poor feed efficiency, and maldigestion. The etiologic agent is a newly identified, but unclassified, virus termed the stunting syndrome agent (SSA). The SSA is a pleomorphic, enveloped virus ranging from 60 to 95 nm in diameter. The objectives of this study were to characterize the physicochemical properties of SSA. SSA hemagglutinated rat erythrocytes at 4 C and room temperature. Treatment of SSA with ether resulted in loss of infectivity. SSA was resistant to pH changes between pH 3.0 and pH 9.0 at 37 C for 1 hr. The virus was inactivated at pH > or = 10. SSA was resistant to treatment with trypsin, chymotrypsin, pancreatin, phospholipase C, and sodium deoxycholate. Treatment of SSA with trypsin, chymotrypsin, and pancreatin resulted in enhanced viral infectivity. The viral genome extracted from purified SSA was sensitive to RNAse treatment. Using oligo d(T)16-18 and random hexamers as primers, the SSA genome was amplified using the reverse transcription-polymerase chain reaction conditions but was not amplified using polymerase chain reaction conditions. The enrichment of viral genome was achieved following poly-A+ selection. These studies provide evidence that the SSA is a positive-sense, single-stranded RNA virus having many characteristics (stability at acidic pH, resistant to proteolytic enzymes and bile salt) consistent with other enveloped enteric viruses.

Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR.

A highly sensitive test based on reverse transcription followed by nested polymerase chain reaction (RT-nPCR) was developed to detect the Australian yellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon. The RT-nPCR detected viral RNA in as little as 10 fg lymphoid organ total RNA isolated from GAV-infected P. monodon. Amplification of serial dilutions of a GAV cDNA clone showed that the nested PCR was sufficiently sensitive to detect a single genome equivalent using a DNA template. The specificity and sensitivity of the RT-nPCR was also demonstrated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified from lymphoid organ and haemocyte RNA as early as 6 h post infection (p.i.), and from gills by 24 h p.i. In contrast, transmission electron microscopy (TEM) identified nucleocapsids and virions in lymphoid organ cells and haemocytes from Days 3 and 6 p.i., respectively, while there was no evidence of infection in gill cells at any time. The practical application of the RT-nPCR was demonstrated by screening healthy wild-caught P. monodon broodstock. The high prevalence (>98%) of broodstock that were positive by RT-nPCR suggests that LOV is endemic in northern Queensland. In addition, results with lymphoid organ, gill and haemocyte RNA suggest that small gill biopsies may be best suited to the non-sacrificial testing of valuable broodstock. The speed and sensitivity of the RT-nPCR make it a useful adjunct to TEM for diagnosing LOV/GAV infection of P. monodon, with the additional benefit that screening of gill biopsies may facilitate selection of LOV-free broodstock.

Bovine coronavirus.

This review aims to summarize current data describing the characteristics of bovine coronavirus (BCV) and the three clinical syndromes with which this virus is associated. The first half of this paper consists of a general description of the virus, commencing with a brief outline of the methods used for in vitro growth. The structure of the virus is then described in more detail, with particular reference to the structure and functions of the four major viral proteins. This is followed by an outline of the unique replication strategy adopted by coronaviruses. The second half of this review discusses the clinical significance of the virus, beginning with a detailed account of BCV-induced neonatal calf diarrhoea, the clinical syndrome with which this virus is most commonly associated. The clinical and epidemiological importance of BCV respiratory tract infection is then discussed, and finally the evidence supporting the aetiological role of BCV in outbreaks of winter dysentery in adult cattle is examined.

Sequence analysis of human coronavirus 229E mRNAs 4 and 5: evidence for polymorphism and homology with myelin basic protein.

Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.

Association of the infectious bronchitis virus 3c protein with the virion envelope.

A highly purified radiolabeled preparation of the coronavirus infectious bronchitis virus (IBV) was analyzed, by immunoprecipitation with monospecific antisera, for the presence of a series of small virus proteins recently identified as the products of IBV mRNAs 3 and 5. One of these, 3c, a 12.4K protein encoded by the third open reading frame of the tricistronic mRNA3 was clearly detectable and was found to cofractionate with virion envelope proteins on detergent disruption of virus particles. These results, together with the hydrophobic nature of 3c and its previously demonstrated association with the membranes of infected cells, suggest strongly that 3c represents a new virion envelope protein, which may have counterparts in other coronaviruses.

Carbohydrate-induced conformational changes strongly modulate the antigenicity of coronavirus TGEV glycoproteins S and M.

The carbohydrate composition and the immunoreactivity of the S and M glycoproteins of the coronavirus TGEV were studied at different stages of their maturation. The biosynthesis of S and M was analyzed in the presence of tunicamycin and monensin. The effect of treatment with endoglycosidases H and F and glycopeptidase F on the precursors and mature forms of S and M were also examined. Species 175K and 29K were characterized as high mannose forms of S and M, respectively, and species 220K and 30-36K as complex type glycosylated forms of these two proteins. M was present mainly as a 29K species in mature virions whereas the 175K form of S was not detected, thus implying that the two proteins undergo Golgi modifications at a far different efficiency. Anti-S and -M monoclonal antibodies were examined for their reactivity towards polypeptide species either treated with endo H or produced in the presence of tunicamycin. It was found that (i) among the four major antigenic sites previously defined (Delmas et al., 1986), only site C (amino acids 363 to 371) was notably expressed by the unglycosylated S polypeptide 155K, whereas the three other sites were dependent upon core-glycosylation, (ii) three of the four anti-M mAbs tested did not recognize the unglycosylated M polypeptide 26K. These data led us to conclude that co-translational, but not terminal glycosylation is an essential requirement for both acquisition and maintenance of the antigenicity of TGEV glycoproteins.