Replicase Genes of Murine Coronavirus Strains A59 and JHM Are Interchangeable: Differences in Pathogenesis Map to the 3' One-Third of the Genome (original) (raw)
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Virology, 1997
C12, an attenuated, fusion delayed, very weakly hepatotropic mutant of mouse hepatitis virus strain A59 (MHV-A59) has been further characterized. We have previously shown that C12 has two amino acid substitutions relative to wild type virus in the spike protein, Q159L (within a region of S1 shown to bind to viral receptor in an in vitro assay) and H716D (in the proteolytic cleavage recognition site). We have sequenced the rest of the 31-kb genome of C12 and compared it to wild type virus. Only three additional amino acids substitutions were found, all encoded within the replicase gene. Analysis of C12 in vivo in C57Bl/6 mice has shown that despite the fact that this virus replicates in the brain to titers at least as high as wild type and causes acute encephalitis similar to wild type, this virus causes a minimal level of demyelination and only at very high levels of virus inoculation. Thus acute encephalitis is not sufficient for the induction of demyelination by MHV-A59. Analysis of mutants isolated at earlier times from the same persistently infected glial cell culture as C12, as well as mutants isolated from a second independent culture of persistently infected glial cells, suggests that both the weakly demyelinating and the weakly hepatotropic phenotypes of C12 are associated with the Q159L amino acid substitution.
Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
PLOS Pathogens, 2005
The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (nonstructural proteins nsp1-nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12-nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II-VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicasetranscriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the nonpermissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive-and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved. Citation: Sawicki SG, Sawicki DL, Younker D, Meyer Y, Thiel V, et al. (2005) Functional and genetic analysis of coronavirus replicase-transcriptase proteins. PLoS Pathog 1(4): e39.
Murine coronavirus neuropathogenesis: determinants of virulence
Journal of NeuroVirology, 2010
Murine coronavirus, mouse hepatitis virus (MHV), causes various diseases depending on the strain and route of inoculation. Both the JHM and A59 strains, when inoculated intracranially or intranasally, are neurovirulent. Comparison of the highly virulent JHM isolate, JHM.SD, with less virulent JHM isolates and with A59 has been used to determine the mechanisms and genes responsible for high neuropathogenicity of MHV. The focus of this review is on the contributions of viral spread, replication, and innate and adaptive immunity to MHV neuropathogenesis. JHM.SD spreads more quickly among neurons than less neurovirulent MHVs, and is able to spread in the absence of the canonical MHV receptor, CEACAM1a. The observation that JHM.SD infects more cells and expresses more antigen, but produces less infectious virus per cell than A59, implies that efficient replication is not always a correlate of high neurovirulence. This is likely due to the unstable nature of the JHM.SD spike protein (S). JHM.SD induces a generally protective innate immune response; however, the strong neutrophil response may be more pathogenic than protective. In addition, JHM.SD induces only a minimal T-cell response, whereas the strong T-cell response and the concomitant interferon-c (IFN-c) induced by the less neurovirulent A59 is protective. Differences in the S and nucleocapsid (N) proteins between A59 and JHM.SD contribute to JHM.SD neuropathogenicity. The hemmagglutinin-esterase (HE) protein may enhance neuropathogenicity of some MHV isolates, but is unlikely a major contributor to the high neuroviruence of JHM.SD. Further data suggest that neither the internal (I) protein nor nonstructural proteins ns4, and ns2 are significant contributors to neurovirulence.
Advances in Virus Research, 2011
A. MHV pathogenesis B. SARS-CoV pathogenesis IV. Coronavirus Reverse Genetics A. Targeted recombination B. Reverse genetic systems that regenerate virus from cDNA V. Structural Proteins A. Spike protein (S) B. Small membrane (E) protein C. Membrane (M) protein D. Hemagglutinin-esterase (HE) E. Nucleocapsid protein (N) and Internal (I) proteins VI. Replicase Proteins A. Nsp12 polymerase and Nsp8 primase B. Nsp13 helicase C. Nsp1 protein D. Nsp3 protein E. Nsp14 protein F. Nsp15 protein G. Nsp16 protein VII. MHV Accessory Proteins A. ns2 protein B. ns4 protein(s) C. ns5a protein
2021
There are many viruses that cause different infectious diseases in human as well as in vertebrate systems of different animals. Coronaviruses are the most infectious viruses belonging to the family Coronaviridae. The genome of coronaviruses are made up of RNA and size about the ~30 kb that contain the 5′ cap structure along with a 3′ poly (A) tail. These are helpful for translating the different proteins causing infections such as replicase proteins. Different types of immune based responses evoked once the viral attack on the immune cells of the body. Accessory proteins also playing important role as inflammatory responses once viral attack on immune cells and release of different inflammatory genes that are involved in progression of COVID-19. One of such kind of response from interferon’s I and III are cytokines that playing important role against the viral infections. Lopinavir and Ritonavir both inhibiting the viral replication and translation by targeting the viral relapse and...
Journal of Neurovirology, 2004
The A59 strain of coronavirus, mouse hepatitis virus (MHV), produces acute hepatitis, meningoencephalitis, and chronic demyelination. The authors have previously shown that the spike (S) glycoprotein gene of MHV contains determinants of virulence, hepatitis, and demyelination. They then identified viruses containing mutations in the S gene that exhibit alterations in viral pathogenesis. In the present study, the authors produced new recombinant viruses with each one of these S gene mutations by site-directed mutagenesis and targeted recombination and studied the effect of each individual mutation on the pathogenesis of the virus. They identified a combination of mutations in the S1 gene (I375M and L652I) that abolishes demyelination. Individual mutation and other combinations of mutations in the S gene only interfere with virulence and hepatitis and only reduce demyelination (I375M), but do not abolish demyelination completely. Thus, demyelination determinants exist within genomic regions on both sides of the hypervariable region, downstream from the receptor-binding domain in the S1 part of the MHV spike glycoprotein gene. The structure and precise function of these regions awaits further investigation.
Journal of Virology, 2005
During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.
Pathogenesis of murine coronavirus in the central nervous system
Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 2010
Murine coronavirus (mouse hepatitis virus, MHV) is a collection of strains that induce disease in several organ systems of mice. Infection with neurotropic strains JHM and A59 causes acute encephalitis, and in survivors, chronic demyelination, the latter of which serves as an animal model for multiple sclerosis. The MHV receptor is a carcinoembryonic antigen-related cell adhesion molecule, CEACAM1a; paradoxically, CEACAM1a is poorly expressed in the central nervous system (CNS), leading to speculation of an additional receptor. Comparison of highly neurovirulent JHM isolates with less virulent variants and the weakly neurovirulent A59 strain, combined with the use of reverse genetics, has allowed mapping of pathogenic properties to individual viral genes. The spike protein, responsible for viral entry, is a major determinant of tropism and virulence. Other viral proteins, both structural and nonstructural, also contribute to pathogenesis in the CNS. Studies of host responses to MHV ...
Virus Research, 1995
The coronavirus spike glycoprotein (S) mediates both the attachment of virus to the host cell receptor and membrane fusion. We describe here the characterization of a temperature-sensitive mutant of the coronavirus mouse hepatitis virus A59 (MHV-A59) having multiple S protein-related defects. The most remarkable of these was that the mutant, designated Albany 18 (Albl8), assembled virions devoid of the S glycoprotein at the nonpermissive temperature. Albl8 also failed to bring about syncytia formation in cells infected at the nonpermissive temperature. Virions of the mutant assembled at the permissive temperature were much more thermolabile than wild type. Moreover, mutant S protein that was incorporated into virions at the permissive temperature showed enhanced pH-dependent thermolability in its ability to bind to the MHV receptor. Albl8 was found to have a single point mutation in S resulting in a change of serine 287 to isoleucin¢, and it was shown by revertant analysis that this was the lesion responsible for the phenotype of the mutant.
Journal of Virology, 2001
The field of replication studies remains a controversial, misunderstood, and unappreciated piñata of 18 replication typologies spanning 79 replication types. To help bring order to the chaos, I contribute a theory of manufactured inferences. The theory is built on three pillars: (1) replication causal diagrams (or r-dags for short), (2) a formal conceptualization of study procedures, and (3) the use of Bayesian inference to update our beliefs about the natural phenomenon under investigation and the operating characteristics of the study procedures used to study it. I use this theory to motivate a formal typology of replication types, explaining how they are done and for what purpose. Finally, I discuss some implications of this theory, including the importance of an analytical approach to robustness and generalizability replications, the need to avoid conceptual replications, the possibility of legitimate (unplanned) specification searches, the limitations of meta-analysis, and the false dichotomy between so-called successful and failed replications.