Recombinant Mouse Hepatitis Virus Research Articles

One proline deletion in the fusion peptide of neurotropic mouse hepatitis virus (MHV) restricts retrograde axonal transport and neurodegeneration

Mouse hepatitis virus (MHV; murine coronavirus) causes meningoencephalitis, myelitis, and optic neuritis followed by axonal loss and demyelination. This murine virus is used as a common model to study acute and chronic virus-induced demyelination in the central nervous system. Studies with recombinant MHV strains that differ in the gene encoding the spike protein have demonstrated that the spike has a role in MHV pathogenesis and retrograde axonal transport. Fusion peptides (FPs) in the spike protein play a key role in MHV pathogenesis. In a previous study of the effect of deleting a single proline residue in the FP of a demyelinating MHV strain, we found that two central, consecutive prolines are important for cell-cell fusion and pathogenesis. The dihedral fluctuation of the FP was shown to be repressed whenever two consecutive prolines were present, in contrast to the presence of a single proline in the chain. Using this proline-deleted MHV strain, here we investigated whether intracranial injection of this strain can induce optic neuritis by retrograde axonal transport from the brain to the retina through the optic nerve. We observed that the proline-deleted recombinant MHV strain is restricted to the optic nerve, is unable to translocate to the retina, and causes only minimal demyelination and no neuronal death. We conclude that an intact proline dyad in the FP of the recombinant demyelinating MHV strain plays a crucial role in translocation of the virus through axons and subsequent neurodegeneration.

Negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus

Coronavirus spike (S) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. Here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of S assembly into mouse hepatitis virus (MHV). Recombinant MHVs exhibited a range of abilities to accommodate the homologous S endodomains from the betacoronaviruses bovine coronavirus and human SARS-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (TGEV), and the gammacoronavirus avian infectious bronchitis virus respectively. Interestingly, in TGEV endodomain chimeras the reverting mutations resulted in stronger S incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. Additionally, MHV S assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. These results indicate an important role for negatively charged endodomain residues in the incorporation of MHV S protein into assembled virions.

Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

Macrophage-Mediated Optic Neuritis Induced by Retrograde Axonal Transport of Spike Gene Recombinant Mouse Hepatitis Virus

After intracranial inoculation, neurovirulent mouse hepatitis virus (MHV) strains induce acute inflammation, demyelination, and axonal loss in the central nervous system. Prior studies using recombinant MHV strains that differ only in the spike gene, which encodes a glycoprotein involved in virus-host cell attachment, demonstrated that spike mediates anterograde axonal transport of virus to the spinal cord. A demyelinating MHV strain induces optic neuritis, but whether this is due to the retrograde axonal transport of viral particles to the retina or due to traumatic disruption of retinal ganglion cell axons during intracranial inoculation is not known. Using recombinant isogenic MHV strains, we examined the ability of recombinant MHV to induce optic neuritis by retrograde spread from the brain through the optic nerve into the eye after intracranial inoculation. Recombinant demyelinating MHV induced macrophage infiltration of optic nerves, demyelination, and axonal loss, whereas optic neuritis and axonal injury were minimal in mice infected with the nondemyelinating MHV strain that differs in the spike gene. Thus, optic neuritis was dependent on a spike glycoprotein-mediated mechanism of viral antigen transport along retinal ganglion cell axons. These data indicate that MHV spreads by retrograde axonal transport to the eye and that targeting spike protein interactions with axonal transport machinery is a potential therapeutic strategy for central nervous system viral infections and associated diseases.

Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus.

The focus of this protocol is mouse hepatitis virus (MHV), with occasional references to other coronaviruses. Many of these protocols can be easily adapted to other coronaviruses. Protocols for propagating MHV in DBT and 17CL‐1 cells; the storage and titration of viral stocks; purification of MHV on sucrose gradients; and the generation of recombinant viruses by a cDNA assembly method and by targeted recombination will be presented. Protocols are also included for the propagation of DBT, 17CL‐1, and L2 cells used for growing and titrating MHV, and for the growth of BHK‐R cells and FCWF cells. The latter two cell lines are used for regenerating infectious MHV by an in vitro cDNA assembly protocol and by a targeted recombination protocol, respectively, allowing reverse genetic manipulation of these viruses. An additional protocol for the maintenance of the large plasmids used for generating recombinant MHVs will also be presented. Curr. Protoc. Microbiol. 21:15E.1.1‐15E.1.46. © 2011 by John Wiley & Sons, Inc.

Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

Deficient incorporation of spike protein into virions contributes to the lack of infectivity following establishment of a persistent, non-productive infection in oligodendroglial cell culture by murine coronavirus

Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.

The Murine Coronavirus Nucleocapsid Gene Is a Determinant of Virulence

The murine coronavirus, mouse hepatitis virus (MHV) strain A59, causes acute encephalitis and chronic demyelinating disease as well as hepatitis in mice. The JHM strain (also called MHV-4 or JHM.SD) causes fatal encephalitis and only minimal hepatitis. Previous analysis of chimeric recombinant MHVs in which the spike gene, encoding the protein that mediates viral entry and cell-to-cell fusion, was exchanged between JHM and A59 showed that the spike plays a major role in determining organ tropism and neurovirulence but that other genes also play important roles in pathogenic outcome. Here, we have investigated the role of the nucleocapsid protein in MHV-induced disease. The multifunctional nucleocapsid protein is complexed with the genomic RNA, interacts with the viral membrane protein during virion assembly, and plays an import role in enhancing the efficiency of transcription. A pair of chimeric recombinant viruses in which the nucleocapsid gene was exchanged between JHM and A59 was selected and compared to wild-type parental strains in terms of virulence. Importantly, expression of the JHM nucleocapsid in the context of the A59 genome conferred increased mortality and spread of viral antigen in the mouse central nervous system compared to the parental A59 strain, while having little effect on the induction of hepatitis. While the JHM nucleocapsid did not appear to enhance neuron-to-neuron spread in primary neuronal cultures, the increased neurovirulence it conferred may be due in part to the induction of a less robust T-cell response than that induced by strain A59.

Insertion of the CXC chemokine ligand 9 (CXCL9) into the mouse hepatitis virus genome results in protection from viral-induced encephalitis and hepatitis

The role of the CXC chemokine ligand 9 (CXCL9) in host defense following infection with mouse hepatitis virus (MHV) was determined. Inoculation of the central nervous system (CNS) of CXCL9−/− mice with MHV resulted in accelerated and increased mortality compared to wild type mice supporting an important role for CXCL9 in anti-viral defense. In addition, infection of RAG1−/− or CXCL9−/− mice with a recombinant MHV expressing CXCL9 (MHV-CXCL9) resulted in protection from disease that correlated with reduced viral titers within the brain and NK cell-mediated protection in the liver. Survival in MHV-CXCL9-infected CXCL9−/− mice was associated with reduced viral burden within the brain that coincided with increased T cell infiltration. Similarly, viral clearance from the livers of MHV-CXCL9-infected mice was accelerated but independent of increased T cell or NK cell infiltration. These observations indicate that CXCL9 promotes protection from coronavirus-induced neurological and liver disease.

Demyelinating and Nondemyelinating Strains of Mouse Hepatitis Virus Differ in Their Neural Cell Tropism

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.

Coronavirus Non-Structural Protein 1 Is a Major Pathogenicity Factor: Implications for the Rational Design of Coronavirus Vaccines

Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.

Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication

All coronaviruses encode a small hydrophobic envelope (E) protein, which mediates viral assembly and morphogenesis by an unknown mechanism. We have previously shown that the E protein from Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) forms cation-selective ion channels in planar lipid bilayers (Wilson, L., McKinlay, C., Gage, P., Ewart, G., 2004. SARS coronavirus E protein forms cation-selective ion channels. Virology 330(1), 322–331). We now report that three other E proteins also form cation-selective ion channels. These E proteins were from coronaviruses representative of taxonomic groups 1–3: human coronavirus 229E (HCoV-229E), mouse hepatitis virus (MHV), and infectious bronchitis virus (IBV), respectively. It appears, therefore, that coronavirus E proteins in general, belong to the virus ion channels family. Hexamethylene amiloride (HMA) – an inhibitor of the HIV-1 Vpu virus ion channel – inhibited the HCoV-229E and MHV E protein ion channel conductance in bilayers and also inhibited replication of the parent coronaviruses in cultured cells, as determined by plaque assay. Conversely, HMA had no antiviral effect on a recombinant MHV with the entire coding region of E protein deleted (MHVΔE). Taken together, the data provide evidence of a link between inhibition of E protein ion channel activity and the antiviral activity of HMA.

Luxury at a Cost? Recombinant Mouse Hepatitis Viruses Expressing the Accessory Hemagglutinin Esterase Protein Display Reduced Fitness In Vitro

Group 2 coronaviruses encode an accessory envelope glycoprotein species, the hemagglutinin esterase (HE), which possesses sialate-O-acetylesterase activity and which, presumably, promotes virus spread and entry in vivo by facilitating reversible virion attachment to O-acetylated sialic acids. While HE may provide a strong selective advantage during natural infection, many laboratory strains of mouse hepatitis virus (MHV) fail to produce the protein. Apparently, their HE genes were inactivated during cell culture adaptation. For this report, we have studied the molecular basis of this phenomenon. By using targeted RNA recombination, we generated isogenic recombinant MHVs which differ exclusively in their expression of HE and produce either the wild-type protein (HE+), an enzymatically inactive HE protein (HE0), or no HE at all. HE expression or the lack thereof did not lead to gross differences in in vitro growth properties. Yet the expression of HE was rapidly lost during serial cell culture passaging. Competition experiments with mixed infections revealed that this was not due to the enzymatic activity: MHVs expressing HE+ or HE0 propagated with equal efficiencies. During the propagation of recombinant MHV-HE+, two types of spontaneous mutants accumulated. One produced an anchorless HE, while the other had a Gly-to-Trp substitution at the predicted C-terminal residue of the HE signal peptide. Neither mutant incorporated HE into virion particles, suggesting that wild-type HE reduces the in vitro propagation efficiency, either at the assembly stage or at a postassembly level. Our findings demonstrate that the expression of "luxury" proteins may come at a fitness penalty. Apparently, under natural conditions the costs of maintaining HE are outweighed by the benefits.

Recombinant Mouse Hepatitis Virus Strain A59 from Cloned, Full-Length cDNA Replicates to High Titers In Vitro and Is Fully Pathogenic In Vivo

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture ( approximately 10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.

Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants.

Both CD4(+) and CD8(+) T cells are required for clearance of the murine coronavirus mouse hepatitis virus (MHV) during acute infection. We investigated the effects of an epitope-specific CD8(+) T-cell response on acute infection of MHV, strain A59, in the murine CNS. Mice with CD8(+) T cells specific for gp33-41 (an H-2D(b)-restricted CD8(+) T-cell epitope derived from lymphocytic choriomeningitis glycoprotein) were infected with a recombinant MHV-A59, also expressing gp33-41, as a fusion protein with enhanced green fluorescent protein (EGFP). By 5 days postinfection, these mice showed significantly (approximately 20-fold) lower titers of infectious virus in the brain compared to control mice. Furthermore mice with gp33-41-specific CD8(+) cells exhibited much reduced levels of viral antigen in the brain as measured by immunohistochemistry using an antibody directed against viral nucleocapsid. More than 90% of the viruses recovered from brain lysates of such protected mice, at 5 days postinfection, had lost the ability to express EGFP and had deletions in their genomes encompassing EGFP and gp33-41. In addition, genomes of viruses from about half the plaques that retained the EGFP gene had mutations within the gp33-41 epitope. On the other hand, gp33-41-specific cells failed to protect perforin-deficient mice from infection by the recombinant MHV expressing gp33, indicating that perforin-mediated mechanisms were needed. Virus recovered from perforin-deficient mice did not exhibit loss of EGFP expression and the gp33-41 epitope. These observations suggest that the cytotoxic T-cell response to gp33-41 exerts a strong immune pressure that quickly selects epitope escape mutants to gp33-41.

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8.

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.

Purified, soluble recombinant mouse hepatitis virus receptor, Bgp1(b), and Bgp2 murine coronavirus receptors differ in mouse hepatitis virus binding and neutralizing activities.

Mouse hepatitis virus receptor (MHVR) is a murine biliary glycoprotein (Bgp1(a)). Purified, soluble MHVR expressed from a recombinant vaccinia virus neutralized the infectivity of the A59 strain of mouse hepatitis virus (MHV-A59) in a concentration-dependent manner. Several anchored murine Bgps in addition to MHVR can also function as MHV-A59 receptors when expressed at high levels in nonmurine cells. To investigate the interactions of these alternative MHVR glycoproteins with MHV, we expressed and purified to apparent homogeneity the extracellular domains of several murine Bgps as soluble, six-histidine-tagged glycoproteins, using a baculovirus expression system. These include MHVR isoforms containing four or two extracellular domains and the corresponding Bgp1(b) glycoproteins from MHV-resistant SJL/J mice, as well as Bgp2 and truncation mutants of MHVR and Bgp1(b) comprised of the first two immunoglobulin-like domains. The soluble four-domain MHVR glycoprotein (sMHVR[1-4]) had fourfold more MHV-A59 neutralizing activity than the corresponding soluble Bgp1(b) (sBgp1(b)) glycoprotein and at least 1,000-fold more neutralizing activity than sBgp2. Although virus binds to the N-terminal domain (domain 1), soluble truncation mutants of MHVR and Bgp1(b) containing only domains 1 and 2 bound virus poorly and had 10- and 300-fold less MHV-A59 neutralizing activity than the corresponding four-domain glycoproteins. In contrast, the soluble MHVR glycoprotein containing domains 1 and 4 (sMHVR[1,4]) had as much neutralizing activity as the four-domain glycoprotein, sMHVR[1-4]. Thus, the virus neutralizing activity of MHVR domain 1 appears to be enhanced by domain 4. The sBgp1(b)[1-4] glycoprotein had 500-fold less neutralizing activity for MHV-JHM than for MHV-A59. Thus, MHV strains with differences in S-glycoprotein sequence, tissue tropism, and virulence can differ in the ability to utilize the various murine Bgps as receptors.

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro.

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

Analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription.

We have inserted heterologous genetic material into the nonessential gene 4 of the coronavirus mouse hepatitis virus (MHV) in order to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the nucleocapsid (N) gene and to develop further genetic tools for site-directed mutagenesis of structural genes other than N. Initially, a 19-nucleotide tag was inserted into the start of gene 4a of MHV strain A59 with the N gene deletion mutant Alb4 as the recipient virus. In further work, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4, creating the currently largest known RNA virus. The expression of GFP was demonstrated by Western blot analysis of infected cell lysates; however, the level of GFP expression was not sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected alteration of the pattern of the nested MHV subgenomic mRNAs. Surprisingly, though, GFP recombinants also produced an RNA species that was the same size as wild-type mRNA4. Analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from 10 different genomic fusion sites, none possessing a canonical intergenic sequence. The finding of these aberrant mRNAs suggests that long-range RNA or the ribonucleoprotein structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.