scholarly journals Genetic Evidence for a Structural Interaction between the Carboxy Termini of the Membrane and Nucleocapsid Proteins of Mouse Hepatitis Virus

2002 ◽  
Vol 76 (10) ◽  
pp. 4987-4999 ◽  
Author(s):  
Lili Kuo ◽  
Paul S. Masters
Keyword(s):  
Hepatitis Virus ◽  
Expression System ◽  
Mouse Hepatitis Virus ◽  
Viral Envelope ◽  
Genetic Evidence ◽  
M Protein ◽  
Structural Protein ◽  
Virion Protein ◽  
Structural Interaction ◽  
Intact Virion

ABSTRACT The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, MΔ2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the MΔ2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.

scholarly journals Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

2000 ◽  
Vol 74 (5) ◽  
pp. 2333-2342 ◽  
Author(s):  
Martin J. B. Raamsman ◽  
Jacomine Krijnse Locker ◽  
Alphons de Hooge ◽  
Antoine A. F. de Vries ◽  
Gareth Griffiths ◽  
...  
Keyword(s):  
Virus Strain ◽  
Hepatitis Virus ◽  
Expression System ◽  
Mouse Hepatitis Virus ◽  
Viral Envelope ◽  
Membrane Structures ◽  
Electron Microscopic ◽  
E Protein ◽  
Infected Cells ◽  
Virus Expression

ABSTRACT The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

scholarly journals Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59

2018 ◽  
Vol 92 (11) ◽  
Author(s):  
Pei Li ◽  
Yiwei Shan ◽  
Wangliang Zheng ◽  
Xiuyuan Ou ◽  
Dan Mi ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Conformational Changes ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
S Protein ◽  
The Stability ◽  
Virus Fitness ◽  
Viral Envelope Proteins

ABSTRACTThe spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness.IMPORTANCEEnveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

scholarly journals Two palmitylated cysteine residues of the severe acute respiratory syndrome coronavirus spike (S) protein are critical for S incorporation into virus-like particles, but not for M–S co-localization

2012 ◽  
Vol 93 (4) ◽  
pp. 823-828 ◽  
Author(s):  
Makoto Ujike ◽  
Cheng Huang ◽  
Kazuya Shirato ◽  
Shutoku Matsuyama ◽  
Shinji Makino ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Immunofluorescence Assay ◽  
M Protein ◽  
Cysteine Residues ◽  
S Protein ◽  
Virion Assembly ◽  
Virus Like Particles ◽  
S Proteins

The endodomain of several coronavirus (CoV) spike (S) proteins contains palmitylated cysteine residues and enables co-localization and interaction with the CoV membrane (M) protein. Depalmitylation of mouse hepatitis virus S proteins abolished this interaction, resulting in the failure of S incorporation into virions. In contrast, an immunofluorescence assay (IFA) showed that depalmitylated severe acute respiratory syndrome coronavirus (SCoV) S proteins still co-localized with the M protein in the budding site. Here, we determined the ability of depalmitylated SCoV S mutants to incorporate S into virus-like particles (VLPs). IFA confirmed that all SCoV S mutants co-localized with the M protein intracellularly. However, the mutants lacking two cysteine residues (C1234/1235) failed to incorporate S into VLPs. This indicated that these palmitylated cysteines are essential for S incorporation, but are not involved in S co-localization mediated by the M protein. Our findings suggest that M–S co-localization and S incorporation occur independently of one another in SCoV virion assembly.

scholarly journals Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

2016 ◽  
Vol 90 (9) ◽  
pp. 4357-4368 ◽  
Author(s):  
Lili Kuo ◽  
Kelley R. Hurst-Hess ◽  
Cheri A. Koetzner ◽  
Paul S. Masters
Keyword(s):  
Nucleocapsid Protein ◽  
Hepatitis Virus ◽  
Virus Assembly ◽  
Mouse Hepatitis Virus ◽  
M Protein ◽  
Growth Defect ◽  
S Protein ◽  
N Protein ◽  
Protein Incorporation ◽  
Carboxy Terminal

ABSTRACTThe coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought.IMPORTANCEThe assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins.

scholarly journals Glycosylation of the Severe Acute Respiratory Syndrome Coronavirus Triple-Spanning Membrane Proteins 3a and M

2006 ◽  
Vol 80 (5) ◽  
pp. 2326-2336 ◽  
Author(s):  
M. Oostra ◽  
C. A. M. de Haan ◽  
R. J. de Groot ◽  
P. J. M. Rottier
Keyword(s):  
Membrane Proteins ◽  
Severe Acute Respiratory Syndrome ◽  
Expression System ◽  
M Protein ◽  
Metabolic Labeling ◽  
Structural Protein ◽  
Reading Frame ◽  
M Proteins ◽  
Group 2 ◽  
Membrane Spanning

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.

scholarly journals The non-structural protein Nsp10 of mouse hepatitis virus binds zinc ions and nucleic acids

FEBS Letters ◽  
2006 ◽  
Vol 580 (17) ◽  
pp. 4143-4149 ◽  
Author(s):  
Nele Matthes ◽  
Jeroen R. Mesters ◽  
Bruno Coutard ◽  
Bruno Canard ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Zinc Ions ◽  
Structural Protein

scholarly journals The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae

2002 ◽  
Vol 83 (2) ◽  
pp. 395-402 ◽  
Author(s):  
Walter Juergen Wurzer ◽  
Karola Obojes ◽  
Reinhard Vlasak
Keyword(s):  
Hepatitis Virus ◽  
Horse Serum ◽  
Specific Interaction ◽  
Mouse Hepatitis Virus ◽  
Alkaline Treatment ◽  
Structural Protein ◽  
Gene Encoding ◽  
Acetylneuraminic Acid ◽  
Group 2 ◽  
Phylogenetic Lineages

Group 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin–esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K m values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, α-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS–polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested.

scholarly journals Crystal Structure of the C-Terminal Cytoplasmic Domain of Non-Structural Protein 4 from Mouse Hepatitis Virus A59

PLoS ONE ◽  
2009 ◽  
Vol 4 (7) ◽  
pp. e6217 ◽  
Author(s):  
Xiaoling Xu ◽  
Zhiyong Lou ◽  
Yanlin Ma ◽  
Xuehui Chen ◽  
Zhangsheng Yang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Cytoplasmic Domain ◽  
Structural Protein
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