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Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evaluated in Crandell feline kidney (CRFK) cells. With a series of bicyclam derivatives, 50% inhibitory concentrations (IC50s) against FIV were obtained in this cell system that were comparable to those obtained for HIV-1 IIIB replication in the human CD4(+) MT-4 T-cell line. The bicyclams were also able to block FIV replication in feline thymocytes, albeit at higher concentrations than in the CRFK cells. The prototype bicyclam AMD3100, 1-1'-[1,4-phenylene-bis(methylene)]-bis(1,4,8, 11-tetraazacyclotetradecane), was only fourfold less active in feline thymocytes (IC50, 62 ng/ml) than in CRFK cells (IC50, 14 ng/ml). AMD2763, 1,1'-propylene-bis(1,4,8, 11-tetraazacyclotetradecane), which is a less potent CXCR4 antagonist, was virtually inactive against FIV in feline thymocytes (IC50, >66.5 microgram/ml), while it was clearly active in CRFK cells (IC50, 0.9 microgram/ml). The CXC chemokine stromal-cell-derived factor 1alpha had anti-FIV activity in CRFK cells (IC50, 200 ng/ml) but not in feline thymocytes (IC50, >2.5 microgram/ml). When primary FIV isolates were evaluated for their drug susceptibility in feline thymocytes, the bicyclams AMD3100 and its Zn2+ complex, AMD3479, inhibited all six primary isolates at equal potency. The marked susceptibility of FIV to the bicyclams suggests that FIV predominantly uses feline CXCR4 for entering its target cells.

Alphaherpesvirus glycoproteins E and I (gE and gI, respectively) assemble into a hetero-oligomeric complex which promotes cell-to-cell transmission, a determining factor of virulence. Focusing on gI of feline herpesvirus (FHV), we examined the role of disulfide bonds during its biosynthesis, its interaction with gE, and gE-gI-mediated spread of the infection in vitro. The protein's disulfide linkage pattern was determined by single and pairwise substitutions for the four conserved cysteine residues in the ectodomain. The resulting mutants were coexpressed with gE in the vaccinia virus-based vTF7-3 system, and the formation and endoplasmic reticulum (ER)-to-Golgi transport of the hetero-oligomeric complex were monitored. The results were corroborated biochemically by performing an endoproteinase Lys-C digestion of a [35S]Cys-labeled secretory recombinant form of gI followed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the peptides under reducing and nonreducing conditions. We found that (i) gI derivatives lacking Cys79 (C1) and/or Cys223 (C4) still assemble with gE into transport-competent complexes, (ii) mutant proteins lacking Cys91 (C2) and/or Cys102 (C3) bind to gE but are retained in the ER, (iii) radiolabeled endoproteinase Lys-C-generated peptide species containing C1 and C4 are linked through disulfide bonds, and (iv) peptides containing both C2 and C3 are not disulfide linked to any other peptide. From these findings emerges a model in which C1 and C4 as well as C2 and C3 form intramolecular disulfide bridges. Since the cysteines in the ectodomain have been conserved during alphaherpesvirus divergence, we postulate that the model applies for all gI proteins. Analysis of an FHV recombinant with a C1-->S substitution confirmed that the C1-C4 disulfide bond is not essential for the formation of a transport-competent gE-gI complex. The mutation affected the posttranslational modification of gI and caused a slight cold-sensitivity defect in the assembly or the intracellular transport of the gE-gI complex but did not affect plaque size. Thus, C1 and the C1-C4 bond are not essential for gE-gI-mediated cell-to-cell spread, at least not in vitro.

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hohdatsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425-433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40-44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5' half of ORF1b of strain 79-1146.

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK(MHVR) cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK(MHVR) cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.

We have characterized the 3'-most 3 kb of the genome of bovine torovirus (BoTV) strain Breda. A novel 1.2-kb gene, located between the genes for the membrane and nucleocapsid proteins, was identified. This gene, the 3'-most 0.5 kb of which is also present in the genome of the equine torovirus isolate Berne virus (BEV), codes for a class I membrane protein displaying 30% sequence identity with the hemagglutinin-esterases (HEs) of coronaviruses and influenza C viruses. Heterologous expression of the BoTV HE gene yielded a 65,000-molecular weight N-glycosylated protein displaying acetylesterase activity. Serologic evidence indicates that the HE homolog is expressed during the natural infection and represents a prominent antigen. By using an antiserum raised against residues 13 to 130 of HE, the HE protein was detected in radioiodinated, sucrose gradient-purified BoTV preparations. Formal evidence that HE is a structural protein was provided by immunoelectron microscopy. In addition to the large, 17- to 20-nm spikes, BoTV virions possess shorter surface projections (6 nm on average). We postulate that these surface projections, which are absent from the BEV virion, are composed of the BoTV HE homolog. The HE gene, which has now been demonstrated in three different virus genera, is a showpiece example of modular evolution.

Cats that have succumbed to feline infectious peritonitis, an immune-mediated disease caused by variants of feline coronaviruses, show apoptosis and T-cell depletion in their lymphoid organs. The ascitic fluid that develops in the course of the condition causes apoptosis in vitro but only in activated T cells. Since feline infectious peritonitis virus does not infect T cells, and viral proteins did not inhibit T-cell proliferation, we postulate that soluble mediators released during the infection cause apoptosis and T-cell depletion.

The biosynthesis of glycoproteins E and I of feline herpesvirus was studied by using the vaccinia virus vTF7-3 expression system. gE and gI were synthesized as N-glycosylated, endoglycosidase H (EndoH)-sensitive precursors with Mrs of 83,000 and 67,000, respectively. When coexpressed, gE and gI formed sodium dodecyl sulfate-sensitive hetero-oligomeric complexes that were readily transported from the endoplasmic reticulum (ER). Concomitantly, the glycoproteins acquired extensive posttranslational modifications, including O glycosylation, leading to an increase in their apparent molecular weights to 95,000 and 80,000 to 100,000 for gE and gI, respectively. In the absence of gE, most gI remained EndoH sensitive. Only a minor population became EndoH resistant, but these molecules were processed aberrantly as indicated by their Mrs (100,000 to 120,000). By immunofluorescence microscopy, gI was detected primarily in the ER but also at the plasma membrane. gE, when expressed by itself, remained EndoH sensitive and was found only in the ER and the nuclear envelope. These results were corroborated by studying the biosynthesis of gE in feline herpesvirus (FHV)-infected cells. In cells infected with wild-type FHV, gE acquired the same co- and posttranslational modifications as during vTF7-3-driven expression. However, an FHV mutant lacking gI failed to produce mature gE. We conclude that gE is retained in the ER, presumably by associating with molecular chaperones, and becomes transport competent only when in a complex with gI.

In a coimmunoprecipitation assay with monospecific antisera, the two major envelope proteins GL and M of equine arteritis virus were found to occur in heteromeric complexes in virions and infected cells. While the GL protein associated with M rapidly and efficiently, newly synthesized M protein was incorporated into complexes at a slower rate, which implies that it interacts with GL molecules synthesized earlier. Analysis under nonreducing conditions revealed that the GL/M complexes consist of disulfide-linked heterodimeric structures. Pulse-chase experiments showed that virtually all GL monomers ended up in heterodimers, whereas a fraction of the M protein persisted as monomers. The M protein also formed covalently linked homodimers, but only the heterodimers were incorporated into virus particles.

Feline immunodeficiency virus (FIV) isolates differ in the ability to replicate in Crandell feline kidney (CRFK) cells. The difference in tropism between two variants of the Dutch isolate FIV-UT113 was studied by using molecular clones which contained the envelope genes of the variants in a background of the FIV-14 Petaluma sequence. Virus produced from clone pPET-113Th replicated in thymocytes, whereas virus from pPET-113Cr propagated in both thymocytes and CRFK cells, thereby reflecting the phenotypes of the parental variants. Exchange of envelope gene fragments showed that a 464-bp surface protein (SU)-encoding fragment encompassing the third variable region (V3) determines CRFK cell tropism. Sequence analysis of the exchanged fragments demonstrated two amino acid changes that led to an increase of the overall charge of the V3 domain: a G-->R transition at position 397 and a E-->K change at position 407. Mutational analysis of these residues revealed that the E-->K shift was responsible for the change in tropism, while the G-->R mutation improved the replication kinetics in CRFK cells. Mapping of a tropism determinant for FIV to a region which is also a major neutralization domain is reminiscent of human immunodeficiency virus type 1, in which a similar colocation was found.

The small membrane glycoprotein (GS) of equine arteritis virus (EAV) is a minor virion component but is abundantly expressed in EAV-infected cells. In this study, we have analyzed its membrane topology, folding, oligomerization, and intracellular transport. We show that GS is a class I integral membrane protein with one functional N-glycosylation site. Gel electrophoresis under nonreducing conditions revealed that GS occurs in EAV-infected cells in four monomeric conformations and as disulfide-linked homodimers. The slowest-migrating monomeric form corresponded to the fully reduced GS protein; the three faster-migrating monomeric species are probably generated by the formation of alternative intrachain disulfide bonds between the three luminal cysteines in the molecule. The GS monomers were selectively retained in the endoplasmic reticulum, as judged by their permanent susceptibility to endoglycosidase H, whereas the GS dimers were specifically incorporated into virus particles and became endoglycosidase H resistant and sialylated during passage through the Golgi apparatus.

The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.

We investigated the role of tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of rat cytomegalovirus (RCMV) infection. TNF-alpha levels found in the sera of radiation-immunosuppressed rats in the course of infection (> 350 pg/ml) correlated with the development of RCMV disease. Administration of anti-TNF-alpha antibodies strongly reduced the severity of pneumonia and led to a reduction in virus titers. In immunocompetent rats, anti-TNF-alpha antibodies also significantly suppressed viral replication. Conversely, administration of TNF-alpha augmented RCMV replication and aggravated the disease signs. In vitro, TNF-alpha enhanced RCMV replication in the macrophage, whereas a reduction of viral replication was observed in fibroblasts, indicating that the effect on viral replication is cell type specific. Besides activation of viral replication and exacerbation of RCMV disease, TNF-alpha also favored lymphoid and hematopoietic tissue reconstitution after irradiation, which may contribute to antiviral resistance and survival. This finding demonstrates the protean nature of TNF-alpha, with both beneficial and adverse effects for the host. Our results suggest that TNF-alpha plays an important role in modulating the pathogenesis of RCMV infection.

We have analyzed the effects of reducing conditions on the folding of the spike (S) protein and on the intracellular transport of the membrane (M) protein of the mouse hepatitis coronavirus. These proteins differ in their potential to form disulfide bonds in the lumen of the endoplasmic reticulum (ER). Intrachain disulfide bonds are formed in the S protein but not in M, which was demonstrated in a pulse-chase experiment by analyzing the viral proteins under nonreducing conditions. To reduce disulfide bonds in vivo, we added dithiothreitol (DTT) to the culture medium of mouse hepatitis coronavirus-infected cells following a procedure recently described by Braakman et al. (I. Braakman, J. Helenius, and A. Helenius, EMBO J. 11:1717-1722, 1992). Short exposure to DTT resulted in the complete reduction of newly synthesized S protein and affected its conformation as judged by the change in mobility in nonreducing gels and by the loss of recognition by a conformation-specific monoclonal antibody. Using this antibody in an immunofluorescence assay, we monitored the reducing effect of DTT in situ. DTT was found to initially affect only the S protein present in the ER; also, after longer treatment, the remaining signal also gradually disappeared. In contrast, folding and transport of the M protein were not inhibited by DTT. Under reducing conditions, M was transported efficiently to the trans side of the Golgi complex, indicating that cellular processes such as ER-to-Golgi transport, O-glycosylation, and Golgi retention were unaffected. In the presence of DTT, the M protein even moved at an increased rate to the Golgi complex, which is probably because of its failure to interact with unfolded S protein. The effects of in vivo reduction were reversible. When DTT was removed from pulse-labeled cells, the S protein folded posttranslationally and aberrantly; during its oxidation, most of S now transiently aggregated into large disulfide-linked complexes from which subsequently folded S molecules dissociated.

We have recently shown that the genome of equine arteritis virus (EAV) contains seven open reading frames (ORFs). We now present data on the structural proteins of EAV and the assignment of their respective genes. Virions are composed of a 14-kDa nucleocapsid protein (N) and three membrane proteins designated M, GS, and GL. M is an unglycosylated protein of 16 kDa, and GS and GL are N-glycosylated proteins of 25 and 30 to 42 kDa, respectively. The broad size distribution of GL results from heterogeneous N-acetyllactosamine addition since it is susceptible to digestion by endo-beta-galactosidase. Using monospecific antisera as well as an antivirion serum, and by expression of individual ORFs, the genes for the structural proteins were identified: ORF 7 codes for N, ORF 6 for M, ORF 5 for GL, and ORF 2 for GS. With the exception of GS, the proteins are about equally abundant in EAV virions, being present at a molar ratio of 3 (N):2 (M):3 (GL). The GS protein, which is expressed at a level similar to that of M in infected cells, is strikingly underrepresented in virus particles (1 to 2%). Our data justify a distinct taxonomic position for EAV, together with lactate dehydrogenase-elevating virus and simian hemorrhagic fever virus; although coronavirus- and toroviruslike in features of transcription and translation, the virion architecture of EAV is fundamentally different.

A new protein of feline infectious peritonitis coronavirus (FIPV) was discovered in lysates of [35S]cysteine-labeled infected cells. Expression of open reading frame (ORF) 6b of FIPV in recombinant vaccinia virus-infected cells was used to identify it as the 6b protein. Further characterization revealed that it is a novel type of viral glycoprotein whose function is not clear. It is a soluble protein contained in microsomes; its slow export from the cell is caused by the presence of an endoplasmic reticulum (ER) retention signal at the C terminus. This amino acid sequence, KTEL, closely resembles the consensus KDEL signal of soluble resident ER proteins. A mutant 6b protein with the C-terminal sequence KTEV became resistant to digestion by endo-beta-N-acetylglucosaminidase H with a half-time that was reduced threefold. In contrast, a mutant with the sequence KDEL was completely retained in the ER. The FIPV 6b protein is the first example of a viral protein with a functional KDEL-like ER retention signal.

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.

The coronavirus spike protein S is responsible for important biological activities including virus neutralization by antibody, cell attachment, and cell fusion. Recently, we have elucidated the amino acid sequence of an S determinant common in murine coronaviruses (W. Luytjes, D. Geerts, W. Posthumus, R. Meloen, and W. Spaan, J. Virol. 63:1408-1412, 1989). A monoclonal antibody directed to this determinant (MAb 5B19.2) protected mice against acute fatal infection. In this study, BALB/c mice were immunized with a synthetic peptide of 13 amino acids corresponding to the binding site of MAb 5B19.2, which was either extended with an amino acid sequence of influenza virus hemagglutinin or conjugated to keyhole limpet hemocyanin. Both immunogens induced S-specific antibodies in mice, but only the hemagglutinin-peptide construct protected them against lethal challenge. In contrast to mouse hepatitis virus type 4 (MHV-4), MHV-A59 was not neutralized in vitro by MAb 5B19.2. Neither MHV-A59 nor MHV-4 was neutralized in vitro by antibodies comprising by the synthetic peptides. Our results demonstrated that antibodies elicited with a synthetic peptide comprising a B-cell epitope and a T-helper cell determinant can protect mice against an acute fetal mouse hepatitis virus infection.

The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis was recombined into the genome of vaccinia virus. The recombinant induced spike-protein-specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with feline infectious peritonitis virus, these animals succumbed earlier than did the control group immunized with wild-type vaccinia virus (early death syndrome).

Coronavirus spike protein genes were expressed in vitro by using the recombinant vaccinia virus expression system. Recombinant spike proteins were expressed at the cell surface and induced cell fusion in a host-cell-dependent fashion. The intracellular transport of recombinant spike proteins was studied. The half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H was approximately 3 h for the recombinant feline infectious peritonitis virus S protein. The S protein in feline infectious peritonitis virus-infected cells was found to have a half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H of approximately 1 h. This difference can be explained by the fact that coronavirus budding takes place at intracellular membranes and that the oligosaccharides of the spike protein are modified after budding. Apparently, spike protein incorporated into budded virions is transported faster through the Golgi apparatus than is spike protein alone. These findings provide new insights into the mechanism of coronavirus budding and are discussed in relation to current models of intracellular transport and sorting of proteins.

By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified.

Three protein species were identified in purified transmissible gastroenteritis virus particles (strain Purdue). They are thought to represent constituents of the peplomer (E2; molecular weights of 280,000 and 240,000), the envelope (E1; molecular weights of 28,000, 31,500, and 33,000), and the nucleocapsid (N; molecular weight of 48,000). In infected cells, proteins with molecular weights of 195,000 (E2), 48,000 (N), and 28,000 (E1) were detected. Tunicamycin, an inhibitor of N glycosylation, prevented the appearance of polypeptides with molecular weights of 195,000 and 28,000 in infected cells; instead, proteins with molecular weights of 160,000 and 25,000 were observed. One minor and five major mRNA species were detected in porcine cells after infection. Their size was determined to be 23.6 kilobases (kb) (RNA1), 8.4 kb (RNA3), 3.8 kb (RNA4), 3.0 kb (RNA5), 2.6 kb (RNA6), and 1.9 kb (RNA7). The RNAs were translated in vitro. RNA7 was shown to code for the N protein. Although complete separation of RNA6 could not be achieved, it was shown to encode an unglycosylated (molecular weight of 25,000) precursor of E1 (molecular weight of 28,000). RNA4 was translated into a nonstructural protein with a molecular weight of 24,000. Translation of RNA3 resulted in proteins with molecular weights of 250,000 and 130,000 and smaller molecules which could be precipitated with a monoclonal antibody directed against E2.

Cell-free cytoplasmic extracts of the Syrian hamster cell lines C13/SV28 and BHK-21F were immunogenic in Syrian hamsters. The resulting antisera cross-reacted completely with antisera against lymphocytic choriomeningitis virus (LCMV) in an immunoradiometric assay employing BHK-21F antigen. Several other Syrian hamster cell lines not previously known to be infected with LCMV were also strongly positive when assayed for viral antigens. Also, several mouse sera and antisera raised in Syrian hamsters against cells transformed by papovaviruses had high titers of anti-LCMV activity. No cytopathic effect was evident in any of the persistently infected cell lines. Culture media from these cells were not infectious and showed no evidence of defective interfering particles. However, cell-free extracts of all the persistently infected cells contained material capable of transmitting the persistent infection to uninfected cells of Syrian hamsters, rats, mice, green monkeys, and humans. The onset of infection is much slower than when LCMV virions are used. When 2 X 10(6) uninfected BHK cells were treated with an extract from 100 persistently infected cells, the new infection was apparent within about 12 days. When an extract from 10(6) cells was used, the new infection was apparent within about 5 days, but not sooner. The intracellular infectious material was sensitive to treatment with deoxycholate, Nonidet P-40, or ether but resistant to treatment with RNase or trypsin. It was also large (5,000S) and heterodisperse on sucrose gradients. The infectious material was probably contained in large lipid vesicles and their integrity was probably essential for infection. When a few persistently infected cells were cocultivated with many uninfected cells, a few discrete colonies positive for LCMV antigens were observed after about 5 days. Since the culture media were not infectious, the infection probably spread by cell-cell contact. Several different experiments indicated that interferon did not play a major role in mediating persistence in this case. Persistent infections by LCMV can be maintained without expression of extracellular virus particles and without appearance of large amounts of viral antigens on the cell surface. Cell-cell contact could still allow transmission of intracellular infectious material. In an animal, these properties could circumvent immune surveillance.

We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by agarose gel electrophoresis of the polyadenylated RNA fraction from infected cells, were obtained pure, except for the preparations of RNAs 4, 5, and 6, which contained some contamination of RNA 7. The RNAs were microinjected into Xenopus laevis oocytes, and after incubation of these cells in the presence of [35S]methionine, the proteins synthesized were analyzed by polyacrylamide gel electrophoresis. Whereas no translation products of RNAs 1, 2, 4, and 5 were detected, the synthesis of virus-specific polypeptides coded by RNAs 3, 6, and 7 was observed. RNA 7 (0.6 X 10(6) daltons) directed the synthesis of a 54,000-molecular-weight polypeptide which comigrated with viral nucleocapsid protein and which was immunoprecipitated by antiserum from mice that had been infected with the virus. RNA 6 (0.9 X 10(6) daltons) directed the synthesis of three polypeptides with molecular weights of 24,000, 25,500, and 26,500, which migrated with the same electrophoretic mobilities as three low-molecular-weight virion polypeptides. After injection of RNA 3 (3.0 X 10(6) daltons), a polypeptide with a molecular weight of about 150,000 was immunoprecipitated. This polypeptide had no counterpart in the virion, but comigrated with a virus-specific glycoprotein present in infected cells which is immunoprecipitated by a rabbit antiserum against the mouse hepatitis virus strain A59 structural proteins. This antiserum could also immunoprecipitate the translation products of RNAs 3, 6, and 7. These results indicate that RNAs 3, 6, and 7 encode viral structural proteins. The significance of the data with respect to the strategy of coronavirus replication is discussed.

Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus.

The target sizes of the templates for the synthesis of the genome-sized RNA and the six subgenomic RNAs found in cells infected with mouse hepatitis virus strain A59 were determined by UV transcription mapping. Infected Sac(-) cells were irradiated at 6 h postinfection, the time when virus-specific RNA synthesis starts to increase exponentially. The effect of increasing UV doses on the synthesis of the individual RNAs was determined by quantitation of these RNAs after separation by agarose gel electrophoresis. The UV target sizes calculated for the templates were almost identical to the physical sizes of the RNAs. The results of these experiments seem to exclude the possibility that the subgenomic RNAs are processed or spliced from a common precursor. The data are consistent with independent initiation of transcription on a genome-sized, negative-stranded template or on smaller templates.

We have shown by T(1) oligonucleotide fingerprinting that the genome of mouse hepatitis virus strain A59 and its intracellular RNA 1 have identical fingerprints and that RNA 1 and the subgenomic RNAs 3, 6, and 7 contain common sequences. To localize the homologous region between the RNAs, we compared fingerprints of the 3' terminus of the genome with those of RNA 7. The genome was partially degraded with alkali, and polyadenylate-containing fragments were purified by oligodeoxythymidylate-cellulose chromatography. The fragments were size fractionated by agarose-urea gel electrophoresis, and two pools, x and z, containing 3'-derived fragments of the genome with apparent molecular weights of 0.1 x 10(6) to 0.14 x 10(6) and 0.6 x 10(6) to 0.8 x 10(6), respectively, were further analyzed by RNase T(1) oligonucleotide fingerprinting. Comparison of the fingerprints of RNAs 6 and 7 with those of pools x and z showed that these subgenomic RNAs extend inwards from the 3' terminus of the genome. The RNA fragments present in pool z were on average slightly larger than RNA 7 as confirmed by the presence in pool z of T(1) oligonucleotide spots specific for RNA 6 but not present in RNA 7. However, two large oligonucleotide spots derived from RNA 7, which were also present in RNAs 1, 3, and 6 and in the virion RNA, were not found in the T(1) oligonucleotide map of pool z. A possible explanation is that the two spots were derived from a leader sequence. The results of UV transcription mapping experiments (L. Jacobs, W. J. M. Spaan, M. C. Horzinek, and B. A. M. van der Zeijst, J. Virol. 39:401-406, 1981) excluded the possibility that such a leader sequence arises by splicing from a larger precursor molecule, but either a virus-specific RNA primer molecule for the synthesis of mRNAs or an RNA polymerase jumping mechanism could explain the presence of a leader sequence.

We identified eight protein species in virions of mouse hepatitis virus strain A59. Based on their sizes, prosthetic groups, and locations in virions, these proteins were designated gp180/E2, gp90/E2, pp54/N, gp26.5/E1, gp25.5/E1, p24/E1, p22/X, and p14.5/Y. The positions of the last two proteins in virions are not known. Host protein synthesis in Sac(-) cells infected with mouse hepatitis virus strain A59 was inhibited, and the following novel proteins appeared: gp150, gp90, p54, gp26.5, gp25.5, p24, p22, and p14.5. Except for gp150, these polypeptides all co-electrophoresed with mouse hepatitis virus strain A59 structural proteins. In addition, all of these proteins could be immunoprecipitated with a convalescent mouse serum or a rabbit antiserum raised against purified disrupted virus. After a 15-min pulse of infected cells with radioactive amino acids at 7h postinfection, gp90 was not detected, whereas gp26.5 and gp25.5 were only labeled to a small extent. During a subsequent chase period gp150 was processed to gp90, whereas the radioactivity in gp26.5 and gp25.5 increased concomitantly with a reduction of label in p24. Tunicamycin, an antibiotic which inhibits the synthesis of glycopeptides bearing N glycosidically linked oligosaccharides, prevented the appearance of gp150 in mouse hepatitis virus strain A59-infected cells. Instead, a 110,000-dalton protein accumulated. In contrast, the syntheses of the smaller viral glycoproteins gp26.5 and gp25.5 were resistant to this drug, indicating that these glycosylations were of the O glycosidical type. Although the production of infectious virus in tunicamycin-treated cells was inhibited by more than 99%, release of noninfectious viral particles continued. An analysis of these particles revealed that they lacked the peplomeric glycoproteins gp90/E2 and gp180/E2. Obviously, although the surface projections were not essential for budding of virus particles from the cells, they were required for infectivity.