Coronavirus Mouse Hepatitis Research Articles

A Murine and a Porcine Coronavirus Are Released from Opposite Surfaces of the Same Epithelial Cells

Epithelial cells are important target cells for coronavirus infection. Earlier we have shown that transmissible gastroenteritis coronavirus (TGEV) and mouse hepatitis coronavirus (MHV) are released from different sides of porcine and murine epithelial cells, respectively. To study the release of these viruses from the same cells, we constructed a porcine LLC-PK1 cell line stably expressing the recombinant MHV receptor cDNA (LMR cells). The MHV and TGEV receptor glycoproteins were shown by immunofluorescence to appear at the surface of the cells and to be functional so that the cells were susceptible to both MHV and TGEV infection. Both coronaviruses entered polarized LMR cells only through the apical surface. Remarkably, while the cells remained susceptible to TGEV for long periods, infectability by MHV decreased with time after plating of the cells onto filters. This was not due to a lack of expression of the MHV receptor, since this glycoprotein was still abundant on the apical surface of these cells. TGEV and MHV appeared to exit LMR cells from opposite sides. Whereas TGEV was released preferentially at the apical membrane, MHV was released preferentially at the basolateral surface. These results show that vesicles containing the two coronaviruses are targeted differently in LMR cells. We propose that the viruses are sorted at the Golgi complex into different transport vesicles that carry information directing them to one of the two surface domains. The apical release of TGEV and the basolateral release of MHV might be factors contributing to the difference in virus spread found between TGEV and MHV in their respective natural hosts, the former causing mainly a localized enteric infection, the latter spreading through the body to other organs.

MHV-A59 Enters Polarized Murine Epithelial Cells through the Apical Surface but Is Released Basolaterally

Coronaviruses have a marked tropism for epithelial cells. Entry and release of the porcine transmissible gastroenteritis virus (TGEV) is restricted to apical surfaces of polarized epithelial cells, as we have recently shown (J. W. A. Rossen, C. P. J. Bekker, W. F. Voorhout, G. J. A. M. Strous, A. van der Ende, and P. J. M. Rottier, 1994, J. Virol. 68, 7966-7973). In this paper we analyze the interactions of mouse hepatitis coronavirus A59 (MHV-A59) with polarized murine kidney cells (mTAL) grown on permeable supports. After inoculation from the apical or basolateral side, virus entry was found to take place only through the apical membrane. The virus utilized a protein of the carcinoembryonic antigen family as its receptor. In contrast to TGEV, MHV-A59 was released preferentially from the basolateral plasma membrane domain, as evidenced by the accumulation of viral proteins and infectivity in the basolateral culture fluid as well as by electron microscopical observations in the mouse, MHV initially replicates in the nasal epithelium before being disseminated throughout the body; the basolateral release of MHV from epithelial cells into the animal's circulation may be the first step in the establishment of a systemic infection.

Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step.

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.

A cis-acting function for the coronavirus leader in defective interfering RNA replication.

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA replication, the wild-type leader is strongly preferred for subsequent replication. They also demonstrate that, in contrast to reported natural mouse hepatitis coronavirus DI RNAs, the DI RNA of the bovine coronavirus does not require sequence elements originating from discontinuous downstream regions within the polymerase gene for replication or for packaging.

Detection of rodent RNA viruses by polymerase chain reaction.

Assays for rodent coronaviruses (mouse hepatitis virus and rat corona/sialodacryoadenitis virus) and a rodent paramyxovirus (Sendai virus) based on the polymerase chain reaction are described.

Mouse hepatitis virus spike and nucleocapsid proteins expressed by adenovirus vectors protect mice against a lethal infection

Infection with the mouse hepatitis coronavirus (MHV) provides an excellent model for the study of viral diseases of the central nervous system and the gastrointestinal tract. With the ultimate aim of studying mucosal immunity to MHV we have cloned the genes encoding the structural proteins of MHV strain A59 (MHV-A59) into the E3 region of a human adenovirus type 5 vector. Infection of HeLa cells with the resulting recombinant adenoviruses AdMHVS, AdMHVN and AdMHVM revealed the correct expression of the spike (S), nucleocapsid (N) and membrane (M) proteins, respectively. Intraperitoneal inoculation of BALB/c mice with the recombinant viruses elicited serum antibodies which specifically recognized the respective MHV proteins in an immunoprecipitation assay. Only antibodies to the S protein neutralized MHV-A59 in vitro but titres were low. When analysed by ELISA or by immunofluorescence only the antibody response to the N protein was significant; weak responses or no detectable response at all were found for S and M, respectively. Upon intracerebral challenge with a lethal dose of MHV-A59 we found that a significant fraction of animals vaccinated with adenovirus vectors expressing either the S protein or N protein were protected. This protective effect was significantly stronger when the animals were given a booster immunization with the same vector prior to challenge. No protection was induced by AdMHVM. Interestingly, enhanced protection resulted when AdMHVS and AdMHVN were applied in combination as compared to survival after single immunizations. The results indicate that both the N and S proteins generate a protective immune response and suggest that this response is enhanced by combined expression of the two proteins.

Leader-mRNA Junction Sequences Are Unique for Each Subgenomic mRNA Species in the Bovine Coronavirus and Remain So Throughout Persistent Infection

The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3′ end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junction region, and in its place was the nonconforming sequence GGTAGAC that occurs just 15 nt downstream in the genome. Leader-mRNA junction sequences found after 296 days of persistent infection were the same as those found during acute infection (<18 hr postinfection). These data indicate that, in contrast to the closely related mouse hepatitis virus, the bovine coronavirus maintains a stable leader-mRNA junction sequence for each mRNA. Interestingly, this stability may be related to the fact that a UCUAA sequence element, postulated by others to be a regulator of the leader-mRNA fusion event, occurs only once within the 3′ flanking sequence of the genomic leader donor and once at intergenic sites in the bovine coronavirus genome, whereas it occurs two to four times at these sites in the mouse hepatitis coronavirus.

Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site

The sequence of the spike (also called peplomer or E2) protein gene of the Mebus strain of bovine coronavirus (BCV) was obtained from cDNA clones of genomic RNA. The gene sequence predicts a 150,825 mol wt apoprotein of 1363 amino acids having an N-terminal hydrophobic signal sequence of 17 amino acids, 19 potential N-linked glycosylation sites, a hydrophobic anchor sequence of approximately 17 amino acids near the C terminus, and a hydrophilic cysteinerich C terminus of 35 amino acids. An internal LysArgArgSerArgArg sequence predicts a protease cleavage site between amino acids 768 and 769 that would separate the S apoprotein into S1 and S2 segments of 85690 and 65153 mol wt, respectively. Amino terminal amino acid sequencing of the virion-derived gp100 spike subunit confirmed the location of the predicted cleavage site, and established that gp120 and gp100 are the glycosylated virion forms of the S1 and S2 subunits, respectively. Sequence comparisons between BCV and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (S1) than in the stem region (S2).

Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59

Complementary DNA (cDNA) libraries were constructed representing the genome RNA of the coronavirus mouse hepatitis virus, strain A59 (MHV-A59). From these libraries clones were selected to form a linear map across the entire gene A, the putative viral polymerase gene. This gene is approximately 23 kb in length, considerably larger than earlier estimates. Sequence analysis of the U terminal region of the genome indicates the presence of the 66-nucleotide leader that is found on all mRNAs. Secondary structure analysis of the 5′terminal region suggests that transcription of leader terminates in the region of nucleotide 66. The sequence of the first 2000 nucleotides is very similar to that reported for the closely related JHM strain of MHV and potentially encodes p28, a basic protein thought to be a component of the viral polymerase (L. Soe, C. K. Shieh, S. Baker, M. F. Chang, and M. M. C. Lai, 1987, J. Virol., 61, 3968–3976) . Gene A contains two of the consensus sequences found in intergenic regions. One is adjacent to the 5′ leader sequence and the other is upstream from the initiation codon for translation of gene B.

The amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation

cDNA clones mapping within the first 2601 bases of the 3′ end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome were sequenced by the method of Maxam and Gilbert and an open reading frame yielding a protein having properties of the matrix (M or E1) protein was identified. It is positioned at the 5′ side of the nucleocapsid (N) gene from which it is separated by an intergenic stretch of 12 bases. The deduced M protein comprises 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has a net charge of +7 at neutral pH. Thirty-four percent of its amino acid sequence is homologous with the M protein of the bovine coronavirus (BCV), 32% with that of the mouse hepatitis coronavirus (MHV), and 19% with that of the avian infectious bronchitis coronavirus (IBV). Judging from alignment with the BCV, MHV, and IBV M proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with only 28 for BCV, 26 for MHV, and 21 for IBV. Eleven of the sixteen amino-terminal amino acids are hydrophobic and the positions of charged amino acids around this sequence suggest that the first 16 amino acids comprise a potentially cleavable signal peptide for membrane insertion. A similar sequence is not found in the M proteins of BCV, MHV, or IBV. When mRNA from infected cells, or RNA prepared by in vitro transcription of the reconstructed M gene, was translated in vitro in the presence of microsomes, the M protein became translocated and glycosylated. When a protein without the amino-terminal signal peptide was made by translating a truncated version of the M gene transcript, some translocation and glycosylation also occurred suggesting that the amino-terminal signal peptide on the TGEV M protein is not an absolute requirement for membrane translocation. Interestingly, the amino-terminal peptide did not appear to be cleaved during in vitro translation in the presence of microsomes suggesting that a step in virion assembly may be required for proper exposure of the cleavage site to the signal peptidase.

Nucleotide sequence of the porcine transmissible gastroenteritis coronavirus matrix protein gene.

cDNA clones mapping within the first 2601 bases of the 3' end of the TGEV genome were sequenced completely or in part by the method of Maxam and Gilbert and open reading frames were examined. One reading frame yielding a protein having properties of the matrix (M) protein was identified. It is positioned at the immediate 5' side of the nucleocapsid (N) gene but is separated by an intergenic region of 12 bases. The deduced M protein is comprised of 262 amino acids, has a molecular weight of 29,544, is moderately hydrophobic, and has an amino acid sequence homology of approximately 36% with the mouse hepatitis coronavirus, 37% with the bovine enteric coronavirus, and 28% with the avian infectious bronchitis virus. Judging from an alignment with MHV and IBV proteins, the amino terminus of the TGEV M protein extends 54 amino acids from the virion envelope which compares with 26 for MHV and 21 for IBV.

Deduced amino acid sequence and potential O-glycosylation sites for the bovine coronavirus matrix protein.

The nucleotide sequence of the matrix (M) protein gene of the bovine coronavirus (BCV) was determined by sequencing cDNA clones derived from genomic RNA. The gene was found to map at the 5' side of the nucleocapsid protein gene and its sequence predicts a protein of 230 amino acids having a molecular weight of 26,376. The BCV M protein shares extensive sequence homology with the matrix protein of the mouse hepatitis coronavirus (MHV) but differs notably in the amino terminal region external to the virion envelope where BCV apparently uses at least two of its six potential O-glycosylation sites.

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene

The 3′ end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into eDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3′ end of the genome. Two potential, nonoverlapping genes were identified within the 3′-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5′ side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3′-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.