Induction of self-reactive T cells after murine coronavirus infection.
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We studied the mechanism of in vitro spontaneous lymphokine production by spleen cells from mice injected intraperitoneally with murine coronavirus stain JHM 1 month after infection, when infectious virus had already been cleared from the spleens. Removal of either CD4+ T cells or Ia+ antigen-presenting cells (APC) from the spleen cells abrogated interleukin-2 (IL-2) production. Addition of anti-CD4 or anti-Iad monoclonal antibodies to the culture suppressed IL-2 production. These results suggest that the response involved typical receptor-mediated activation of T cells. Surprisingly, reciprocal mixing experiments with a coculture of T cells from infected mice and APC from either infected or naive mice resulted in the production of IL-2. The absence of viral antigens in spleen cells 1 month after infection, as indicated by their inability to induce the proliferation of T-cell clones specific for the viral antigens, suggest that the T cells from mice 1 month after infection were not responding to the viral antigens. The inoculum components other than the virus did not induce this immune response. We also found that the frequency of self-reactive but not alloreactive IL-2-producing T cells in the spleens of infected mice was 3- to 10-fold higher than that in naive mice. These findings suggest that an increased frequency of self-reactive T cells which secrete IL-2 occurs following murine coronavirus infection. This may have important implications in the development of autoimmunelike phenomena following murine coronavirus infection. (+info)
Alteration of the pH dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein.
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Infection of susceptible murine cells with the coronavirus mouse hepatitis virus type 4 (MHV4) results in extensive cell-cell fusion at pHs from 5.5 to 8.5. The endosomotropic weak bases chloroquine and ammonium chloride do not prevent MHV4 infection. In marked contrast, we have selected variants from a neural cell line persistently infected with MHV4 which are entirely dependent on acid pH to fuse host cells and are strongly inhibited by endosomotropic weak bases. Wild-type and variant viruses were compared at the level of the fusion-active surface (S) glycoprotein gene. Cloning and sequencing of each 4,131-base open reading frame predicted a total of eight amino acid differences which fell into three distinct clusters. Each S glycoprotein, when expressed from cDNA, was synthesized in equivalent amounts, and similar proportions were transported to the cell surface. Wild-type S induced cell-cell fusion at neutral pH, whereas variant S required prolonged exposure to acidic pH to induce fusion. Expression of hybrid S genes prepared by exchange of restriction fragments between wild-type and variant cDNAs revealed that elimination of neutral pH fusion was solely dependent on amino acid alterations at positions 1067 (Q to H), 1094 (Q to H), and 1114 (L to R). These changes lie within a predicted heptad repeat region of the transmembrane cleavage fragment of S (S2). These findings demonstrate that the pH dependence of coronavirus fusion is highly variable and that this variability can be determined by as few as three amino acid residues. (+info)
Sequence comparison of the 5' end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus.
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Analysis of porcine transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) mRNA species indicated a deletion in mRNA 3 of PRCV. Polymerase chain reaction (PCR) was used to clone the 5' end of mRNA 3 from PRCV for comparison with the equivalent region in TGEV. Small deletions were observed within and around the PRCV sequence equivalent to the putative open reading frame (ORF) ORF-3a identified in TGEV. The potential RNA polymerase-leader complex binding site (leader RNA binding site), ACTAAAC, found upstream of ORF-3a in TGEV, was absent from the PRCV genome but a potential site was found in the PRCV genome upstream of a gene equivalent to TGEV ORF-3b. PCR analysis, using primers corresponding to sequences within the ORF-3b gene and the leader RNA sequence, confirmed that the leader RNA binding site was upstream of a gene equivalent to TGEV ORF-3b on PRCV mRNA 3 but upstream of ORF-3a on TGEV mRNA 3. The presence of the new leader RNA binding site would be responsible for generating the smaller mRNA 3 species found in PRCV-infected cells. (+info)
Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.
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The nucleotide sequence of the genome of equine arteritis virus (EAV) was determined from a set of overlapping cDNA clones and was found to contain eight open reading frames (ORFs). ORFs 2 through 7 are expressed from six 3'-coterminal subgenomic mRNAs, which are transcribed from the 3'-terminal quarter of the viral genome. A number of these ORFs are predicted to encode structural EAV proteins. The organization and expression of the 3' part of the EAV genome are remarkably similar to those of coronaviruses and toroviruses. The 5'-terminal three-quarters of the genome contain the putative EAV polymerase gene, which also shares a number of features with the corresponding gene of corona- and toroviruses. The gene contains two large ORFs, ORF1a and ORF1b, with an overlap region of 19 nucleotides. The presence of a "shifty" heptanucleotide sequence in this region and a downstream RNA pseudoknot structure indicate that ORF1b is probably expressed by ribosomal frameshifting. The frameshift-directing potential of the ORF1a/ORF1b overlap region was demonstrated by using a reporter gene. Moreover, the predicted ORF1b product was found to contain four domains which have been identified in the same relative positions in coronavirus and torovirus ORF1b products. The sequences of the EAV and coronavirus ORF1a proteins were found to be much more diverged. The EAV ORF1a product contains a putative trypsinlike serine protease motif. Our data indicate that EAV, presently considered a togavirus, is evolutionarily related to viruses from the coronaviruslike superfamily. (+info)
Genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus.
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The genome and transcriptional pattern of a newly identified respiratory variant of transmissible gastroenteritis virus were analyzed and compared with those of classical enterotropic transmissible gastroenteritis virus. The transcriptional patterns of the two viruses indicated that differences occurred in RNAs 1 and 2(S) and that RNA 3 was absent in the porcine respiratory coronavirus (PRCV) variant. The smaller RNA 2(S) of PRCV was due to a 681-nucleotide (nt) deletion after base 62 of the PRCV peplomer or spike (S) gene. The PRCV S gene still retained information for the 16-amino-acid signal peptide and the first 6 amino acid residues at the N terminus of the mature S protein, but the adjacent 227 residues were deleted. Two additional deletions (3 and 5 nt) were detected in the PRCV genome downstream of the S gene. The 3-nt deletion occurred in a noncoding region; however, the 5-nt deletion shortened the potential open reading frame A polypeptide from 72 to 53 amino acid residues. Significantly, a C-to-T substitution was detected in the last base position of the transcription recognition sequence upstream of open reading frame A, which rendered RNA 3 nondetectable in PRCV-infected cell cultures. (+info)
Sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus.
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The 3' end of the turkey coronavirus (TCV) genome and the gene encoding the nucleocapsid protein (N) were cloned and sequenced. The gene encoding the membrane protein (M) was obtained by cloning a polymerase chain reaction (PCR)-amplified fragment obtained using bovine coronavirus (BCV)-specific primers. Furthermore, five TCV DNA fragments, obtained by PCR on RNA from clinical specimens and corresponding to either the N terminus of the M protein or the complete M protein were also cloned and sequenced. The sequence revealed a 3' non-coding region of 291 bases, an open reading frame (ORF) encoding the N protein with a predicted size of 448 amino acids, or an Mr of 49K, and an ORF encoding the M protein with a predicted size of 230 amino acids and an Mr of 26K. A third ORF, encoding a hypothetical protein of 207 amino acids with an Mr of 23K was found within the N gene sequence. The amino acid sequences of both the N and M proteins were more than 99% similar to those published for BCV. Extensive similarity was also observed between the amino acid sequences of the TCV N protein and those of murine hepatitis virus (MHV) (70%) and human respiratory coronavirus strain OC43 (HCV-OC43) (98%) and between the amino acid sequences of the predicted M proteins of TCV and MHV (86%). Such striking identity suggests that BCV, TCV and HCV-OC43 must have diverged from each other only recently. A potential N-glycosylation site was found at the N terminus of the TCV M protein and is situated at the same location in BCV, MHV and transmissible gastroenteritis virus. (+info)
The Golgi sorting domain of coronavirus E1 protein.
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The coronavirus E1 membrane protein is confined to the Golgi after it is expressed in cells either by viral infection or via injection of synthetic RNA. We have investigated the features of the protein responsible for intracellular sorting and found that a C-terminal deletion of only 18 amino acids results in its transport to the plasma membrane. However, we have previously shown that this C-terminal region alone is not sufficient for Golgi retention. When E1 was fused to a cell-surface protein, Thy-1, the resulting molecule was retained in the Golgi. Various mutated forms of E1 whose destinations were the ER, cell surface or lysosomes were also fused to Thy-1, and in each case the fusion was sorted according to its E1 component alone. We argue that, in contrast to sorting signals for other membrane compartments, Golgi retention of E1 is not due to a single short peptide sequence. Instead, the Golgi 'signal' of E1 appears to require for its expression a domain comprising most of the sequence of the protein. (+info)
The detection of transmissible gastroenteritis viral antibodies by immunodiffusion.
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Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of othet viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroeneritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus. (+info)