Targeted Recombination within the Spike Gene of Murine Coronavirus Mouse Hepatitis Virus-A59: Q159 Is a Determinant of Hepatotropism

Previous studies of a group of mutants of the murine coronavirus mouse hepatitis virus (MHV)-A59, isolated from persistently infected glial cells, have shown a strong correlation between a Q159L amino acid substitution in the S1 subunit of the spike gene and a loss in the ability to induce hepatitis and demyelination. To determine if Q159L alone is sufficient to cause these altered pathogenic properties, targeted RNA recombination was used to introduce a Q159L amino acid substitution into the spike gene of MHV-A59. Recombination was carried out between the genome of a temperature-sensitive mutant of MHV-A59 (Alb4) and RNA transcribed from a plasmid (pFV1) containing the spike gene as well as downstream regions, through the 3′ end, of the MHV-A59 genome. We have selected and characterized two recombinant viruses containing Q159L. These recombinant viruses (159R36 and 159R40) replicate in the brains of C57BL/6 mice and induce encephalitis to a similar extent as wild-type MHV-A59. However, they exhibit a markedly reduced ability to replicate in the liver or produce hepatitis compared to wild-type MHV-A59. These viruses also exhibit reduced virulence and reduced demyelination. A recombinant virus containing the wild-type MHV-A59 spike gene, wtR10, behaved essentially like wild-type MHV-A59. This is the first report of the isolation of recombinant viruses containing a site-directed mutation, encoding an amino acid substitution, within the spike gene of any coronavirus. This technology will allow us to begin to map the molecular determinants of pathogenesis within the spike glycoprotein.     

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by approximately 100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an approximately 5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.     

Molecular characterization of feline infectious peritonitis virus strain DF-2 and studies of the role of ORF3abc in viral cell tropism

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log(10) titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.     

Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV(BAC)) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV(Luc)). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV(Luc) genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.     

Pathogenesis of Chimeric MHV4/MHV-A59 Recombinant Viruses: the Murine Coronavirus Spike Protein Is a Major Determinant of Neurovirulence

The mouse hepatitis virus (MHV) spike glycoprotein, S, has been implicated as a major determinant of viral pathogenesis. In the absence of a full-length molecular clone, however, it has been difficult to address the role of individual viral genes in pathogenesis. By using targeted RNA recombination to introduce the S gene of MHV4, a highly neurovirulent strain, into the genome of MHV-A59, a mildly neurovirulent strain, we have been able to directly address the role of the S gene in neurovirulence. In cell culture, the recombinants containing the MHV4 S gene, S4R22 and S4R21, exhibited a small-plaque phenotype and replicated to low levels, similar to wild-type MHV4. Intracranial inoculation of C57BL/6 mice with S4R22 and S4R21 revealed a marked alteration in pathogenesis. Relative to wild-type control recombinant viruses (wtR13 and wtR9), containing the MHV-A59 S gene, the MHV4 S gene recombinants exhibited a dramatic increase in virulence and an increase in both viral antigen staining and inflammation in the central nervous system. There was not, however, an increase in the level of viral replication in the brain. These studies demonstrate that the MHV4 S gene alone is sufficient to confer a highly neurovirulent phenotype to a recombinant virus deriving the remainder of its genome from a mildly neurovirulent virus, MHV-A59. This definitively confirms previous findings, suggesting that the spike is a major determinant of pathogenesis.     

The spike glycoprotein of murine coronavirus MHV-JHM mediates receptor-independent infection and spread in the central nervous systems of Ceacam1a-/- Mice

The MHV-JHM strain of the murine coronavirus mouse hepatitis virus is much more neurovirulent than the MHV-A59 strain, although both strains use murine CEACAM1a (mCEACAM1a) as the receptor to infect murine cells. We previously showed that Ceacam1a^−/− mice are completely resistant to MHV-A59 infection (E. Hemmila et al., J. Virol. 78:10156-10165, 2004). In vitro, MHV-JHM, but not MHV-A59, can spread from infected murine cells to cells that lack mCEACAM1a, a phenomenon called receptor-independent spread. To determine whether MHV-JHM could infect and spread in the brain independent of mCEACAM1a, we inoculated Ceacam1a^−/− mice. Although Ceacam1a^−/− mice were completely resistant to i.c. inoculation with 10⁶ PFU of recombinant wild-type MHV-A59 (RA59) virus, these mice were killed by recombinant MHV-JHM (RJHM) and a chimeric virus containing the spike of MHV-JHM in the MHV-A59 genome (SJHM/RA59). Immunohistochemistry showed that RJHM and SJHM/RA59 infected all neural cell types and induced severe microgliosis in both Ceacam1a^−/− and wild-type mice. For RJHM, the 50% lethal dose (LD₅₀) is <10^1.3 in wild-type mice and 10^3.1 in Ceacam1a^−/− mice. For SJHM/RA59, the LD₅₀ is <10^1.3 in wild-type mice and 10^3.6 in Ceacam1a^−/− mice. This study shows that infection and spread of MHV-JHM in the brain are dependent upon the viral spike glycoprotein. RJHM can initiate infection in the brains of Ceacam1a^−/− mice, but expression of mCEACAM1a increases susceptibility to infection. The spread of infection in the brain is mCEACAM1a independent. Thus, the ability of the MHV-JHM spike to mediate mCEACAM1a-independent spread in the brain is likely an important factor in the severe neurovirulence of MHV-JHM in wild-type mice.     

Mouse hepatitis virus liver pathology is dependent on ADP-ribose-1'-phosphatase, a viral function conserved in the alpha-like supergroup

Viral infection of the liver can lead to severe tissue damage when high levels of viral replication and spread in the organ are coupled with strong induction of inflammatory responses. Here we report an unexpected correlation between the expression of a functional X domain encoded by the hepatotropic mouse hepatitis virus strain A59 (MHV-A59), the high-level production of inflammatory cytokines, and the induction of acute viral hepatitis in mice. X-domain (also called macro domain) proteins possess poly-ADP-ribose binding and/or ADP-ribose-1′′-phosphatase (ADRP) activity. They are conserved in coronaviruses and in members of the “alpha-like supergroup” of phylogenetically related positive-strand RNA viruses that includes viruses of medical importance, such as rubella virus and hepatitis E virus. By using reverse genetics, we constructed a recombinant murine coronavirus MHV-A59 mutant encoding a single-amino-acid substitution of a strictly conserved residue that is essential for coronaviral ADRP activity. We found that the mutant virus replicated to slightly reduced titers in livers but, strikingly, did not induce liver disease. In vitro, the mutant virus induced only low levels of the inflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6). In vivo, we found that IL-6 production, in particular, was reduced in the spleens and livers of mutant virus-infected mice. Collectively, our data demonstrate that the MHV X domain exacerbates MHV-induced liver pathology, most likely through the induction of excessive inflammatory cytokine expression.     

The Spike Protein of Murine Coronavirus Mouse Hepatitis Virus Strain A59 Is Not Cleaved in Primary Glial Cells and Primary Hepatocytes

Mouse hepatitis virus strain A59 (MHV-A59) produces meningoencephalitis and severe hepatitis during acute infection. Infection of primary cells derived from the central nervous system (CNS) and liver was examined to analyze the interaction of virus with individual cell types derived from the two principal sites of viral replication in vivo. In glial cell cultures derived from C57BL/6 mice, MHV-A59 produces a productive but nonlytic infection, with no evidence of cell-to-cell fusion. In contrast, in continuously cultured cells, this virus produces a lytic infection with extensive formation of syncytia. The observation of few and delayed syncytia following MHV-A59 infection of hepatocytes more closely resembles infection of glial cells than that of continuously cultured cell lines. For MHV-A59, lack of syncytium formation correlates with lack of cleavage of the fusion glycoprotein, or spike (S) protein. The absence of cell-to-cell fusion following infection of both primary cell types prompted us to examine the cleavage of the spike protein. Cleavage of S protein was below the level of detection by Western blot analysis in MHV-A59-infected hepatocytes and glial cells. Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59. Thus, cleavage of the spike protein does not seem to be essential for entry and spread of the virus in vivo, as well as for replication in vitro.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Sequence to structural analysis of ORF5 protein in Norway rat Hepatitis E Virus

Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein’s function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/β structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.     

Interaction of Kaposi's sarcoma-associated herpesvirus ORF59 with oriLyt is dependent on binding with K-Rta

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) displays two distinct life stages, latency and lytic reactivation. Progression through the lytic cycle and replication of the viral genome constitute an essential step toward the production of infectious virus and human disease. KSHV K-RTA has been shown to be the major transactivator required for the initiation of lytic reactivation. In the transient-cotransfection replication assay, K-Rta is the only noncore protein required for DNA synthesis. K-Rta was shown to interact with both C/EBPα binding motifs and the R response elements (RRE) within oriLyt. It is postulated that K-Rta acts in part to facilitate the recruitment of replication factors to oriLyt. In order to define the role of K-Rta in the initiation of lytic DNA synthesis, we show an interaction with ORF59, the DNA polymerase processivity factor (PF), one of the eight virally encoded proteins necessary for origin-dependent DNA replication. Using the chromatin immunoprecipitation (ChIP) assay, both K-Rta and ORF59 interact with the RRE and C/EBPα binding motifs within oriLyt in cells harboring the KSHV bacterial artificial chromosome (BAC). A transient-transfection ChIP assay demonstrated that the interaction of ORF59 with oriLyt is dependent on binding with K-Rta and that ORF59 fails to bind to oriLyt in the absence of K-Rta. Also, using the cotransfection replication assay, overexpression of the interaction domain of K-Rta with ORF59 has a dominant negative effect on oriLyt amplification, suggesting that the interaction of K-Rta with ORF59 is essential for DNA synthesis and supporting the hypothesis that K-Rta facilitates the formation of a replication complex at oriLyt.     

Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus.

The defective interfering (DI) RNA MIDI of mouse hepatitis virus strain A59 (MHV-A59) contains a large open reading frame (ORF) spanning almost its entire genome. This ORF consists of sequences derived from ORF1a, ORF1b, and the nucleocapsid gene. We have previously demonstrated that mutations that disrupt the ORF decrease the fitness of MIDI and its derivatives (R. J. de Groot, R. G. van der Most, and W. J. M. Spaan, J. Virol. 66:5898-5905, 1992). To determine whether translation of the ORF per se is required or whether the encoded polypeptide or a specific sequence is involved, we analyzed sets of related DI RNAs containing different ORFs. After partial deletion of ORF1b and nucleocapsid gene sequences, disruption of the remaining ORF is still lethal; translation of the entire ORF is not essential, however. When a large fragment of the MHV-A59 spike gene, which is not present in any of the MHV-A59 DI RNAs identified so far, was inserted in-frame into a MIDI derivative, translation across this sequence was vital to DI RNA survival. Thus, the translated sequence is irrelevant, indicating that translation per se plays a crucial role in DI virus propagation. Next, it was examined during which step of the viral life cycle translation plays its role. Since the requirement for translation also exists in DI RNA-transfected and MHV-infected cells, it follows that either the synthesis or degradation of DI RNAs is affected by translation.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two-open reading frame 3a (ORF3a) and ORF7a-have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a -1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.     

Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells

Murine hepatitis virus (strain A59), (MHV-A59) is a coronavirus that buds into pre-Golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. The fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. MHV-A59 also infects, albeit inefficiently, AtT20 cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. Here we examine AtT20 cells at early times after the infection, when the Golgi apparatus retains its morphological and biochemical integrity. We observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same Golgi stacks and accumulate in the trans-Golgi network. Their pathways diverge at this site, the condensed secretory proteins including the ACTH going to the secretory granules and the coronavirus to post-Golgi transport vesicles devoid of ACTH. On very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-Golgi vesicles. We conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein ACTH, diverge at the exit from the trans-Golgi network.     

The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation

The organ tropism of MHV-A59, a murine coronavirus, was studied in 4-6 week-old C57BL/6 mice inoculated by different routes and with various amounts of virus. MHV-A59 caused hepatitis after intracerebral and intraperitoneal inoculation (two clearly artificial routes) and also after intranasal and intragastric inoculation (two routes more likely to mimic naturally acquired infection). For each route, the severity of hepatitis was dependent on the amount of virus inoculated. Significantly higher doses were needed to cause hepatitis by the intranasal or intragastric routes. We have shown previously that mice inoculated intracerebrally with MHV-A59 develop mild meningoencephalitis followed by chronic central nervous system (CNS) disease, characterized by primary demyelination (1). We extend these results here to show that acute CNS disease can be produced also by intranasal and intragastric inoculation, although much larger doses are needed as compared to intracerebral inoculation. Thus induction of demyelination, not only by the intracerebral route but also by the intranasal route, provides a useful model system to study virus-induced demyelination.