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Development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2008; 16:241-5. [PMID: 19038782 DOI: 10.1128/cvi.00252-08] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
A new enzyme-linked immunosorbent assay (ELISA)-based immunoglobulin G (IgG)-plus-IgM antibody detection test for severe acute respiratory syndrome (SARS) has been developed by using a cocktail of four recombinant polypeptides as the antigen. These recombinant fragments were designed as parts of two different structural proteins from SARS-associated coronavirus (SARS-CoV). One recombinant polypeptide, S251-683, was designed as part of the spike glycoprotein, and the other three polypeptides comprised almost the whole nucleocapsid protein, avoiding the last 25 C-terminal amino acids. Immunization with a cocktail of these four polypeptides yielded a specific polyclonal antibody that is able to recognize SARS-CoV-infected cells by an immunofluorescence assay. This polypeptide cocktail was also used to set up an ELISA-based IgG-plus-IgM antibody detection test, which showed 99% specificity and 90% sensitivity upon evaluation using sera from 100 healthy negative controls and 20 SARS patients. Separate immunoreactivity assays with each recombinant polypeptide demonstrated that a combination of N and S protein fragments was more suitable than the individual peptides for developing a serological assay for SARS-CoV.
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Liu RS, Yang KY, Lin J, Lin YW, Zhang ZH, Zhang J, Xia NS. High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris. World J Gastroenterol 2004; 10:3602-7. [PMID: 15534914 PMCID: PMC4612000 DOI: 10.3748/wjg.v10.i24.3602] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein. This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.
METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His-Mut+) by electroporation. His+Mut+ recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.
RESULTS: All of the recombinants were His+Mut+ after transformation of P.pastoris with linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains. The final optimal concentration of methanol was 20 mL/L, the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved, and a maximum cell A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum. The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in E.coli when the same rSCoVN protein quantity was used.
CONCLUSION: Active recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (rSCoVN) protein can be successfully expressed in recombinant methylotrophic yeast P.pastoris GS115. The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum. This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.
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Affiliation(s)
- Ru-Shi Liu
- The Key Laboratory for Cell Biology and Tumor Cell Engineering of the Ministry of Education; The Research Center for Medical Molecular Virology of Fujian Province, Xiamen University, Xiamen 361005, Fujian Province, China
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Manopo I, Lu L, He Q, Chee LL, Chan SW, Kwang J. Evaluation of a safe and sensitive Spike protein-based immunofluorescence assay for the detection of antibody responses to SARS-CoV. J Immunol Methods 2004; 296:37-44. [PMID: 15680149 PMCID: PMC7094334 DOI: 10.1016/j.jim.2004.10.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2004] [Revised: 06/17/2004] [Accepted: 10/22/2004] [Indexed: 11/29/2022]
Abstract
Previously, we have identified a truncated antigenic fragment named protein C [441 to 700 amino acids (a.a.)] as the immunodominant fragment of Spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We have now successfully expressed protein C using the baculovirus system in S. frugiperda (Sf-9) cells. This recombinant baculovirus expressing protein C was first characterized using five SARS convalescent human sera and five normal human sera. The results showed that protein C is an authentic antigen against SARS-CoV antibody. Our Spike protein-based immunoflourescence assay (IFA) based on this recombinant baculovirus-Sf-9 system was further assessed with a panel of 163 clinical samples collected during the SARS epidemic in Singapore, which include samples from 21 clinically confirmed SARS, 42 non-SARS patient sera, and 100 normal sera. The results were compared to a commercial SARS IFA kit (EUROIMMUN, Germany) and a conventional IFA test performed in Singapore General Hospital. All of the 21 SARS-positive serum samples could be recognized by our IFA, giving a specificity and sensitivity of 100%, which was compatible with both whole virus-based IFA assays. No cross-reactivity with serum samples against infectious bronchitis virus (IBV) and transmissible gastroenteritis virus (TGEV) were detected in our assays. Thus, our Spike protein-based IFA could offer a safer procedure which can be performed in a BSL-2 laboratory as it could mimic the whole virus based-IFA without any loss of sensitivity and specificity. It is also more user-friendly and cost-effective than the whole virus-based IFA.
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Affiliation(s)
| | | | | | | | | | - Jimmy Kwang
- Corresponding author. Tel.: +65 68727473; fax: +65 68727007.
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Lu L, Manopo I, Leung BP, Chng HH, Ling AE, Chee LL, Ooi EE, Chan SW, Kwang J. Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus. J Clin Microbiol 2004; 42:1570-6. [PMID: 15071006 PMCID: PMC387621 DOI: 10.1128/jcm.42.4.1570-1576.2004] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.
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Affiliation(s)
- Liqun Lu
- Animal Health Biotechnology Unit, Temasek Life Science Laboratory, National University of Singapore
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5
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Ruan Y, Wei CL, Ling AE, Vega VB, Thoreau H, Se Thoe SY, Chia JM, Ng P, Chiu KP, Lim L, Zhang T, Chan KP, Lin Ean LO, Ng ML, Leo SY, Ng LFP, Ren EC, Stanton LW, Long PM, Liu ET. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection. Lancet 2003; 361:1779-85. [PMID: 12781537 PMCID: PMC7140172 DOI: 10.1016/s0140-6736(03)13414-9] [Citation(s) in RCA: 331] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines. METHODS We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04). FINDINGS We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing. INTERPRETATION Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations.
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Affiliation(s)
- Yijun Ruan
- Genome Institute of Singapore, Singapore
| | | | - Ai Ee Ling
- Virology Section, Department of Pathology, Singapore General Hospital, Singapore
| | | | | | - Su Yun Se Thoe
- Virology Section, Department of Pathology, Singapore General Hospital, Singapore
| | | | - Patrick Ng
- Genome Institute of Singapore, Singapore
| | | | - Landri Lim
- Genome Institute of Singapore, Singapore
| | - Tao Zhang
- Genome Institute of Singapore, Singapore
| | - Kwai Peng Chan
- Virology Section, Department of Pathology, Singapore General Hospital, Singapore
| | - Lynette Oon Lin Ean
- Virology Section, Department of Pathology, Singapore General Hospital, Singapore
| | - Mah Lee Ng
- Department of Microbiology and Electron Microscopy Unit, National University of Singapore
| | | | - Lisa FP Ng
- Genome Institute of Singapore, Singapore
| | | | | | | | - Edison T Liu
- Genome Institute of Singapore, Singapore
- Correspondence to: Dr Edison T Liu, 1 Science Park Road 05–01, Singapore Science Park II, Singapore, 117528
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Yamada YK, Yabe M, Ohtsuki T, Taguchi F. Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site. Virus Res 2000; 66:149-54. [PMID: 10725548 PMCID: PMC7125849 DOI: 10.1016/s0168-1702(99)00134-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The membrane (M) proteins of murine coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser-Ser-Thr-Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.
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Affiliation(s)
- Y K Yamada
- Division of Experimental Animal Research, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo, Japan.
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Yamada YK, Yabe M. Sequence analysis of major structural proteins of newly isolated mouse hepatitis virus. Exp Anim 2000; 49:61-6. [PMID: 10803365 DOI: 10.1538/expanim.49.61] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022] Open
Abstract
We have isolated the virus from a fecal pellet in the colon of a BALB/c mouse with X-linked immunodeficiency (xid) housed in a room in which there has recently been an epidemic due to mouse hepatitis virus (MHV) and designated it as the MHV-TY strain. Sequence analysis of the MHV-TY strain was performed on major structural, spike (S), membrane (M) and nucleocapsid (N), proteins directly from PCR products. The comparison of nucleotide sequences of MHV-TY with other strains investigated so far revealed that all three structural proteins of the TY strain had some unique amino acid sequences among MHV strains which can be used as markers of this strain.
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Affiliation(s)
- Y K Yamada
- Division of Experimental Animal Research, National Institute of Infectious Diseases, Tokyo, Japan
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de Haan CA, Roestenberg P, de Wit M, de Vries AA, Nilsson T, Vennema H, Rottier PJ. Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein. J Biol Chem 1998; 273:29905-14. [PMID: 9792708 DOI: 10.1074/jbc.273.45.29905] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The mouse hepatitis virus (MHV) membrane (M) protein contains only O-linked oligosaccharides. We have used this protein as a model to study the structural requirements for O-glycosylation. We show that MHV M is modified by the addition of a single oligosaccharide side chain at the cluster of 4 hydroxylamino acids present at its extreme amino terminus and identified Thr at position 5 as the functional acceptor site. The hydroxylamino acid cluster, which is quite conserved among O-glycosylated coronavirus M proteins, is not in itself sufficient for O-glycosylation. Downstream amino acids are required to introduce a functional O-glycosylation site into a foreign protein. In a mutagenic analysis O-glycosylation was found to be sensitive to some particular changes but no unique sequence motif for O-glycosylation could be identified. Expression of mutant M proteins in cells revealed that substitution of any 1 residue was tolerated, conceivably due to the occurrence of multiple UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situ glycosylation assay based on the co-expression of endoplasmic reticulum-retained forms of the GalNAc transferases with endoplasmic reticulum-resident MHV M mutants. The GalNAc transferases were found to have largely overlapping, but distinct substrate specificities. The requirement for a threonine as acceptor rather than a serine residue and the requirement for a proline residue three positions downstream of the acceptor site were found to be distinctive features.
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Affiliation(s)
- C A de Haan
- Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and the Institute of Biomembranes, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands
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9
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de Haan CA, Kuo L, Masters PS, Vennema H, Rottier PJ. Coronavirus particle assembly: primary structure requirements of the membrane protein. J Virol 1998; 72:6838-50. [PMID: 9658133 PMCID: PMC109893 DOI: 10.1128/jvi.72.8.6838-6850.1998] [Citation(s) in RCA: 196] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.
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Affiliation(s)
- C A de Haan
- Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands
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Morita E, Ebina H, Muto A, Himeno H, Hatakeyama K, Sugiyama K. Primary structures of hemagglutinin-esterase and spike glycoproteins of murine coronavirus DVIM. Virus Genes 1998; 17:123-8. [PMID: 9857985 PMCID: PMC7089163 DOI: 10.1023/a:1008060522426] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Diarrhea virus of infant mice (DVIM) is a member of murine hepatitis viruses (MHVs). The nucleotide sequences of the genes encoding the hemagglutinin-esterase (HE) and the spike (S) glycoproteins from DVIM were determined and compared with those of other MHVs. The deduced amino acid sequence of the HE protein was most similar to that of MHV-S strain (94% identity), and the S protein sequence was most similar to that of MHV-Y strain (90% identity). The DVIM HE protein has a unique N-linked glycosylation site in addition to other glycosylation sites common to many MHV strains. Unlike in some typical MHV strain, such as MHV-A59 and MHV-JHM, the vast majority of the S glycoprotein molecules in DVIM exist an uncleaved form probably due to several amino acid substitutions around the cleavage site.
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Affiliation(s)
- E Morita
- Department of Biology, Faculty of Science, Hirosaki University, Japan
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Woods RD. Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates. CANADIAN JOURNAL OF VETERINARY RESEARCH = REVUE CANADIENNE DE RECHERCHE VETERINAIRE 1997; 61:167-72. [PMID: 9242995 PMCID: PMC1189399] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Sixteen isolates of transmissible gastroenteritis virus and one isolate of porcine respiratory coronavirus were characterized using RT-PCR amplification of 4 antigenic subsites in the site A epitope on the TGEV spike gene. The PCR products were digested with restriction enzymes Sau3AI and SspI and the sizes of the fragments were determined. Three different digestion patterns were observed with each enzyme. The recognition site for Sau3AI was missing in 1 isolate, was present in 13 isolates and 3 isolates had 2 sites. PCR-products with a single site had 3 different fragment sizes and the other isolates produced 2 fragments with different sizes. The SspI recognition site was not present in 5 isolates and 12 isolates had a single site that produced 2 fragments of different sizes. Based on the restriction fragment sizes, the 17 isolates were separated into 7 groups. Direct sequencing of the 455 bp nested set fragments demonstrated greater than 96% sequence homology among the 16 isolates and 100% homology in the 4 antigenic subsites in the conserved site A epitope. The groups are discussed in relation to their sequence homology and virulence. In vitro procedures have been developed to identify several porcine enteric coronavirus isolates at the strain level.
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Affiliation(s)
- R D Woods
- Virology Swine Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa 50010, USA
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12
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Kunita S, Zhang L, Homberger FR, Compton SR. Molecular characterization of the S proteins of two enterotropic murine coronavirus strains. Virus Res 1995; 35:277-89. [PMID: 7785316 PMCID: PMC7134003 DOI: 10.1016/0168-1702(94)00089-u] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Enterotropic strains of murine coronaviruses (MHV-Y and MHV-RI) differ extensively in their pathogenesis from the prototypic respiratory strains of murine coronaviruses. In an effort to determine which viral proteins might be determinants of enterotropism, immunoblots of MHV-Y and MHV-RI virions using anti-S, -N and -M protein-specific antisera were performed. The uncleaved MHV-Y and MHV-RI S proteins migrated slightly faster than the MHV-A59 S protein. The MHV-Y S protein was inefficiently cleaved. The MHV-Y, MHV-RI and MHV-A59 N and M proteins showed only minor differences in their migration. The S genes of MHV-Y and MHV-RI were cloned, sequenced and found to encode 1361 and 1376 amino acid long proteins, respectively. The presence of several amino acids changes upstream from the predicted cleavage site of the MHV-Y S protein may contribute its inefficient cleavage. A high degree of homology was found between the MHV-RI and MHV-4 S proteins, whereas the homology between the MHV-Y S protein and the S proteins of other MHV strains was much lower. These results indicate that the enterotropism of MHV-RI and MHV-Y may be determined by different amino acid changes in the S protein and/or by changes in other viral proteins.
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Affiliation(s)
- S Kunita
- Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520-8016, USA
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13
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Homberger FR. Sequence analysis of the nucleoprotein genes of three enterotropic strains of murine coronavirus. Arch Virol 1995; 140:571-9. [PMID: 7733827 PMCID: PMC7087234 DOI: 10.1007/bf01718432] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/1994] [Accepted: 11/15/1994] [Indexed: 01/26/2023]
Abstract
The nucleotide sequences of the nucleoprotein genes of three enterotropic strains of the murine coronavirus mouse hepatitis virus (MHV-Y, MHV-RI and DVIM) were determined and compared with previously reported sequences of three polytropic (respiratory) strains (MHV-A59, MHV-JHM and MHV-S). Greater than 92% homology was found among the six strains by pair-wise comparison at the nucleotide level. The genes encoded proteins of 451 to 455 residues and the deduced amino acid sequences were more than 91% homologous. A unique deletion of twelve nucleotides was found at the carboxy terminus of MHV-Y and a three nucleotide deletion was found in MHV-RI, which corresponded to the one previously reported in MHV-A59 and MHV-S. Two internal open reading frames were found within the coding region of the nucleoprotein, the smaller one was specific for the enterotropic strains. It could potentially encode a truncated version of the hypothetical protein described for MHV-A59 and MHV-S. Sequence relationship of the N gene showed no correlation with tissue tropism and no sequence or even single amino acid change unique to either tropism group was found. This indicates that the nucleoprotein of MHV probably has no part in the determination of the primary tissue tropism of an MHV strain. The role of the potential internal protein warrants further investigation.
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Affiliation(s)
- F R Homberger
- Institute of Laboratory Animal Science, University of Zurich, Switzerland
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