Attenuation of Mouse Hepatitis Virus by Deletion of the LLRKxGxKG Region of Nsp1

Coronaviruses are a family of large positive-sense RNA viruses that are responsible for a wide range of important veterinary and human diseases. Nsp1 has been shown to have an important role in the pathogenetic mechanisms of coronaviruses in vivo. To assess the function of a relatively conserved domain (LLRKxGxKG) of MHV nsp1, a mutant virus, MHV-nsp1-27D, with a 27 nts (LLRKxGxKG) deletion in nsp1, was constructed using a reverse genetic system with a vaccinia virus vector. The mutant virus had similar growth kinetics to MHV-A59 wild-type virus in 17CI-1 cells, but was highly attenuated in vivo. Moreover, the mutant virus completely protected C57BL/6 mice from a lethal MHV-A59 challenge. To further analyze the mechanism of the attenuation of the mutant virus, changes in reporter gene expression were measured in nsp1- or nsp1-27D-expressing cells; the results showed that nsp1 inhibited reporter gene expression controlled by different promoters, but that this inhibition was reduced for nsp1-27D. The research in vivo and in vitro suggests that the LLRKxGxKG region of nsp1 may play an important role in this process.     

Single-amino-acid substitutions in open reading frame (ORF) 1b-nsp14 and ORF 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice

A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The "in vitro-assembled" wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log10 50% lethal dose (LD50) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log10 LD5), and the "corrected" Tyr6398/Leu94 virus had a log10 LD50 identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.     

PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production

Infections by coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus (SCoV) and mouse hepatitis virus A59 (MHV-A59) result in very little type I interferon (IFN) production by host cells, which is potentially responsible for the rapid viral growth and severe immunopathology associated with SARS. However, the molecular mechanisms for the low IFN production in cells infected with coronaviruses remain unclear. Here, we provide evidence that Papain-like protease domain 2 (PLP2), a catalytic domain of the nonstructural protein 3 (nsp3) of MHV-A59, can bind to IRF3, cause its deubiquitination and prevent its nuclear translocation. As a consequence, co-expression of PLP2 strongly inhibits CARDIF-, TBK1- and IRF3-mediated IFNbeta reporter activities. In addition, we show that wild-type PLP2 but not the mutant PLP2 lacking the deubiquitinase (DUB) activity can reduce IFN induction and promote viral growth in cells infected with VSV. Thus, our study uncovered a viral DUB which coronaviruses may use to escape from the host innate antiviral responses.     

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.     

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Induces Apoptosis Dependent on Its 3C-Like Serine Protease Activity

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by PRRS virus (PRRSV). Although PRRSV infection-induced cell apoptosis has been established, the related viral protein is still unknown. Here, we reported that PRRSV nonstructural protein 4 (nsp4) was a critical apoptosis inducer. Nsp4 could activate caspase-3, -8, and -9. Using truncated constructs without different domains in nsp4, we demonstrated that the full-length of nsp4 structure was required for its apoptosis-inducing activity. Furthermore, using site-directed mutagenesis to inactivate the 3C-like serine protease activity of nsp4, we showed that nsp4-induced apoptosis was dependent on its serine protease activity. The ability of nsp4 to induce apoptosis was significantly impaired by His39, Asp64, and Ser118 mutations, suggesting that His39, Asp64, and Ser118 were essential for nsp4 to trigger apoptosis. In conclusion, our present work showed that PRRSV nsp4 could induce apoptosis in host cells and might be partially responsible for the apoptosis induced by PRRSV infection. PRRSV 3C-like protease-mediated apoptosis represents the first report in the genus Arterivirus, family Arteriviridae.     

Crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn2+-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 A. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by approximately 120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.     

The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity.

The spike protein (S) of the murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) induces both virus-to-cell fusion during infection and syncytium formation. Thus far, only syncytium formation could be studied after transient expression of S. We have recently described a system in which viral infectivity is mimicked by using virus-like particles (VLPs) and reporter defective-interfering (DI) RNAs (E. C. W. Bos, W. Luytjes, H. Van der Meulen, H. K. Koerten, and W. J. M. Spaan, Virology 218:52-60, 1996). Production of VLPs of MHV-A59 was shown to be dependent on the expression of M and E. We now show in several ways that the infectivity of VLPs is dependent on S. Infectivity was lost when spikeless VLPs were produced. Infectivity was blocked upon treatment of the VLPs with MHV-A59-neutralizing anti-S monoclonal antibody (MAb) A2.3 but not with nonneutralizing anti-S MAb A1.4. When the target cells were incubated with antireceptor MAb CC1, which blocks MHV-A59 infection, VLPs did not infect the target cells. Thus, S-mediated VLP infectivity resembles MHV-A59 infectivity. The system can be used to identify domains in S that are essential for infectivity. As a first application, we investigated the requirements of cleavage of S for the infectivity of MHV-A59. We inserted three mutant S proteins that were previously shown to be uncleaved (E. C. W. Bos, L. Heijnen, W. Luytjes, and W. J. M. Spaan, Virology 214:453-463, 1995) into the VLPs. Here we show that cleavage of the spike protein of MHV-A59 is not required for infectivity.     

Purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis virus (MHV)-A59 from mouse liver and identification of a nonfunctional, homologous protein in MHV-resistant SJL/J mice.

The receptor for mouse hepatitis virus strain A59 (MHV-A59) is a 110- to 120-kilodalton (kDa) glycoprotein which is expressed in MHV-susceptible mouse strains on the membranes of hepatocytes, intestinal epithelial cells, and macrophages. SJL/J mice, which are highly resistant to MHV-A59, were previously shown to lack detectable levels of receptor by using either solid-phase virus receptor assays or binding of a monoclonal anti-receptor antibody (MAb) which blocks infection of MHV-susceptible mouse cells. This MAb was used for affinity purification of the receptor glycoprotein from livers of MHV-susceptible Swiss Webster mice. The MHV receptor and an antigenically related protein of 48 to 58 kDa were copurified and then separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 15 amino acids of the receptor were sequenced, and a synthetic peptide of this amino acid sequence was prepared. Rabbit antiserum made against this peptide bound to the MHV receptor glycoprotein and the 48- to 58-kDa protein from livers of MHV-susceptible BALB/c mice and Swiss Webster mice and from the intestinal brush border of BALB/c mice. In immunoblots of intestinal brush border and hepatocyte membranes of MHV-resistant SJL/J mice, the antibody against the amino terminus of the receptor identified proteins that are 5 to 10 kDa smaller than the MHV receptor and the 48- to 58-kDa related protein from Swiss Webster or BALB/c mice. Thus, SJL/J mice express a protein which shares some sequence homology with the MHV receptor but which lacks virus-binding activity and is not recognized by the blocking anti-receptor MAb. These results suggest that resistance of SJL/J mice to MHV-A59 may be due to absence or mutation of the virus-binding domain in the nonfunctional receptor homolog in SJL/J mice.     

Effects of interleukin 17A (IL-17A) neutralization on murine hepatitis virus (MHV-A59) infection

Mice infected with mouse hepatitis virus A59 (MHV-A59) develop hepatitis and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH), a fact closely related to the release of alarmins such as uric acid and/or high-mobility group box protein 1 (HMGB1). We studied the effect of neutralizing monoclonal antibodies (MAb) against IL-17A in our model of mouse MHV-A59-infection. MAb anti-IL-17F and anti-IFNγ were used to complement the study. Results showed that transaminase levels markedly decreased in MHV-A59-infected mice treated with MAb anti-IL-17A whereas plasmatic Ig concentration sharply increased. Conversely, MAb anti-IL-17F enhanced transaminase liberation and did not affect Ig levels. Serum IFNγ was detected in mice infected with MHV-A59 and its concentration increased after MAb anti-IL-17A administration. Besides, MAb anti-IFNγ greatly augmented transaminase plasmatic levels. IL-17A neutralization did not affect MHV-A59-induction of HMGB1 liberation and slightly augmented plasmatic uric acid concentration. However, mice treated with the MAb failed to produce autoAb to FAH. The above results suggest a reciprocal regulation of Th1 and Th17 cells acting on the different MHV-A59 effects. In addition, it is proposed that IL-17A is involved in alarmins adjuvant effects leading to autoAb expression.     

Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle

The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.     

东部马脑脊髓炎病毒的分子生物学进展

东部马脑脊髓炎病毒属虫媒病毒,能引起人和马发生急性脑炎。东马病毒为单股正链RNA病毒,可分为南美型和北美型,包括两个开放读码框架,分别编码结构蛋白（E1,E2,E2,C,6K）和非结构蛋白（nsp1,nsp2,nsp3,nsp4)。其中E1/E2包膜糖蛋白以异二聚体的形式病毒颗粒外刺突。非结构蛋白主要能与负链RNA的合成,近来,随着研究深入,病毒受体越来越受到广泛关注。本介绍东部马脑脊髓炎病毒结构、进化、复制、组装等方面的分子生物学进展。     

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 murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.     

Structural consequences of the B5 histidine --> tyrosine mutation in human insulin characterized by X-ray crystallography and conformational analysis.

The addition of phenols to hexameric insulin solutions produces a particularly stable hexamer, resulting from a rearrangement in which residues B1-B8 change from an extended conformation (T-state) to form an alpha-helix (R-state). The R-state is, in part, stabilized by nonpolar interactions between the phenolic molecule and residue B5 His at the dimer-dimer interface. The B5 His --> Tyr mutant human insulin was constructed to see if the tyrosine side chain would mimic the effect of phenol binding in the hexamer and induce the R-state. In partial support of this hypothesis, the molecule crystallized as a half-helical hexamer (T(3)R(3)) in conditions that conventionally promote the fully nonhelical (T6) form. As expected, in the presence of phenol or resorcinol, the B5 Tyr hexamers adopt the fully helical (R6) conformation. Molecular modeling calculations were performed to investigate the conformational preference of the T-state B5 Tyr side chain in the T(3)R(3) form, this side chain being associated with structural perturbations of the A7-A10 loop in an adjacent hexamer. For an isolated dimer, several different orientations of the side chain were found, which were close in energy and readily interconvertible. In the crystal environment only one of these conformations remains low in energy; this conformation corresponds to that observed in the crystal structure. This suggests that packing constraints around residue B5 Tyr result in the observed structural rearrangements. Thus, rather than promoting the R-state in a manner analogous to phenol, the mutation appears to destabilize the T-state. These studies highlight the role of B5 His in determining hexamer conformation and in mediating crystal packing interactions, properties that are likely be important in vivo.