TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium.
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Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity.
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The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E. (+info)
Development and application of an enzyme immunoassay for coronavirus OC43 antibody in acute respiratory illness.
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Study of coronavirus OC43 infections has been limited because of the lack of sensitive cell culture systems and serologic assays. To improve this circumstance, we developed an indirect enzyme immunoassay (EIA) to detect serum antibody to OC43. Antigen (100 ng) prepared by polyethylene glycol precipitation provided optimal results without a postcoat procedure. Evaluation of intraplate variation indicated that a > or = 2.5-fold increase in serum titer was significant. Sixteen of 18 (89%) paired serum samples with previously identified, reproducible increases in the level of hemagglutination inhibition (HAI) antibody to OC43 also showed significant increases as detected by EIA. Specificity for the EIA was established with paired sera obtained from persons given influenza immunizations or experiencing a respiratory infection. No rise in antibody titers occurred among 33 persons with documented coronavirus 229E infection. EIA was then performed on each of 419 paired serum samples from ambulatory chronic obstructive pulmonary disease patients and healthy older adults, from asthmatic adults presenting for emergency room treatment, and from persons hospitalized with acute respiratory symptoms. Twenty-three antibody rises to OC43 were detected; only nine of these were detected by the HAI test, and the HAI test did not detect any increases in antibody titers that were not detected by EIA. Nineteen of 25 coronavirus OC43 infections for which a month of infection could be assigned occurred between November and February. Overall, 4.4% of acute respiratory illnesses in the studied populations were associated with a coronavirus OC43 infection. (+info)
Nucleotide sequence and expression of the spike (S) gene of canine coronavirus and comparison with the S proteins of feline and porcine coronaviruses.
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We have cloned, sequenced and expressed the spike (S) gene of canine coronavirus (CCV; strain K378). Its deduced amino acid sequence has revealed features in common with other coronavirus S proteins: a stretch of hydrophobic amino acids at the amino terminus (the putative signal sequence), another hydrophobic region at the carboxy terminus (the membrane anchor), heptad repeats preceding the anchor, and a cysteine-rich region located just downstream from it. Like other representatives of the same antigenic cluster (CCV-Insavc-1 strain, feline infectious peritonitis and enteric coronaviruses, porcine transmissible gastroenteritis and respiratory coronaviruses, and the human coronavirus HCV 229E), the CCV S polypeptide lacks a proteolytic cleavage site present in many other coronavirus S proteins. Pairwise comparisons of the S amino acid sequences within the antigenic cluster demonstrated that the two CCV strains (K378 and Insavc-1) are 93.3% identical, about as similar to each other as they are to the two feline coronaviruses. The porcine sequences are clearly more divergent mainly due to the large differences in the amino-terminal (residues 1 to 300) domains of the proteins; when only the carboxy-terminal parts (residues 301 and on) are considered the homologies between the canine, feline and porcine S polypeptides are generally quite high, with identities ranging from 90.8% to 96.8% . The human coronavirus is less related to the other members of the antigenic group. A phylogenetic tree constructed on the basis of the S sequences showed that the two CCVs are evolutionarily more related to the feline than to the porcine viruses. Expression of the CCV S gene using the vaccinia virus T7 RNA polymerase system yielded a protein of the expected M(r) (approximately 200K) which could be immunoprecipitated with an anti-feline infectious peritonitis virus polyclonal serum and which was indistinguishable from the S protein synthesized in CCV-infected cells. (+info)
An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA.
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The RNA polymerase gene (gene 1) of the human coronavirus 229E is approximately 20 kb in length and is located at the 5' end of the positive-strand genomic RNA. The coding sequence of gene 1 is divided into two large open reading frames, ORF1a and ORF1b, that overlap by 43 nucleotides. In the region of the ORF1a/ORF1b overlap, the genomic RNA displays two elements that are known to mediate (-1) ribosomal frameshifting. These are the slippery sequence, UUUAAAC, and a 3' pseudoknot structure. By introducing site-specific mutations into synthetic mRNAs, we have analysed the predicted structure of the HCV 229E pseudoknot and shown that besides the well-known stem structures, S1 and S2, a third stem structure, S3, is required for a high frequency of frameshifting. The requirement for an S3 stem is independent of the length of loop 2. (+info)
Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E.
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Gene 1 of the human coronavirus HCV 229E encompasses approximately 20.7 kb and contains two overlapping open reading frames, ORF 1a and ORF 1b. The downstream ORF 1b is expressed by a mechanism involving (-1) ribosomal frameshifting. Translation of mRNA 1, which is thought to be equivalent to the viral genomic RNA, results in the synthesis of two large polyproteins, pp1a and pp1ab. These polyproteins contain motifs characteristic of papain-like and 3C-like proteinases, RNA-dependent RNA polymerases, helicases, and metal-binding proteins. In this study, we have produced pp1ab-specific monoclonal antibodies and have used them to detect an intracellular, 105-kDa viral polypeptide that contains the putative RNA polymerase domain. Furthermore, using trans cleavage assays with bacterially expressed HCV 229E 3C-like proteinase, we have demonstrated that the 105-kDa polypeptide is released from pp1ab by cleavage at the dipeptide bonds Gln-4068/Ser-4069 and Gln-4995/Ala-4996. These data contribute to the characterization of coronavirus 3C-like proteinase-mediated processing of pp1ab and provide the first identification of an HCV 229E ORF 1ab-encoded polypeptide in virus-infected cells. (+info)