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 Functional analysis of an epitope in the S2 subunit of the murine coronavirus spike protein: involvement in fusion activity

2000 ◽  
Vol 81 (12) ◽  
pp. 2867-2871 ◽  
Author(s):  
Fumihiro Taguchi ◽  
Yohko K. Shimazaki
Keyword(s):  
Monoclonal Antibody ◽  
Hepatitis Virus ◽  
Virus Entry ◽  
Mouse Hepatitis Virus ◽  
Spike Protein ◽  
Fusion Activity ◽  
S Protein ◽  
Fusion Inhibition ◽  
Hydrophobic Amino Acids ◽  
S Proteins

The monoclonal antibody (MAb) 5B19.2, which has virus-neutralizing and fusion inhibition activities, binds to an epitope (S2A) consisting of nine hydrophobic amino acids in the S2 subunit of the mouse hepatitis virus (MHV) spike (S) protein. This suggests that the S2A epitope may be involved in binding the virus to the MHV receptor and/or in virus–cell fusion. Co-immunoprecipitation analyses demonstrated that while the binding of virus to the receptor was blocked by anti-S1 MAbs, it was not blocked by the S2A antiserum, indicating that S2A was not involved in receptor-binding. The S proteins prepared in this study with mutations in the S2A epitope were either fusogenic or non-fusogenic and their fusogenicity did not correlate with the hydrophobic feature of the S2A epitope. All of these wt and mutated S proteins were similarly transported onto the cell membrane independent of their fusogenicity capability. These results suggest that S2A may mediate the fusion activity of the MHV S protein during virus entry into cells.

scholarly journals Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH

2019 ◽  
Vol 93 (20) ◽  
Author(s):  
Dan Mi ◽  
Xiuyuan Ou ◽  
Pei Li ◽  
Guiqing Peng ◽  
Yan Liu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Hepatitis Virus ◽  
Virus Entry ◽  
Mouse Hepatitis Virus ◽  
S Protein ◽  
High Ph ◽  
Terminal Domain ◽  
S Proteins

ABSTRACT Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696–10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered. IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the β1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.

scholarly journals Envelope glycoprotein interactions in coronavirus assembly.

1995 ◽  
Vol 131 (2) ◽  
pp. 339-349 ◽  
Author(s):  
D J Opstelten ◽  
M J Raamsman ◽  
K Wolfs ◽  
M C Horzinek ◽  
P J Rottier
Keyword(s):  
Envelope Glycoprotein ◽  
Hepatitis Virus ◽  
Virus Assembly ◽  
Higher Order ◽  
M Protein ◽  
Lag Phase ◽  
S Protein ◽  
Viral Membrane ◽  
Golgi Membranes ◽  
S Proteins

Coronaviruses are assembled by budding into smooth membranes of the intermediate ER-to-Golgi compartment. We have studied the association of the viral membrane glycoproteins M and S in the formation of the virion envelope. Using coimmunoprecipitation analysis we demonstrated that the M and S proteins of mouse hepatitis virus (MHV) interact specifically forming heteromultimeric complexes in infected cells. These could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (NP-40) and ionic (deoxycholic acid) detergents proved to be optimal. Pulse-chase experiments revealed that newly made M and S proteins engaged in complex formation with different kinetics. Whereas the M protein appeared in complexes immediately after its synthesis, newly synthesized S protein did so only after a lag phase of > 20 min. Newly made M was incorporated into virus particles faster than S, which suggests that it associates with preexisting S molecules. Using the vaccinia virus T7-driven coexpression of M and S we also demonstrate formation of M/S complexes in the absence of other coronaviral proteins. Pulse-chase labelings and coimmunoprecipitation analyses revealed that M and S associate in pre-Golgi membranes because the unglycosylated form of M appeared in M/S complexes rapidly. Since no association of M and S was detected when protein export from the ER was blocked by brefeldin A, stable complexes most likely arise in the ER-to-Golgi intermediate compartment. Sucrose velocity gradient analysis showed the M/S complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo- and heterotypic interactions. M/S complexes colocalized with alpha-mannosidase II, a resident Golgi protein. They acquired Golgi-specific oligosaccharide modifications but were not detected at the cell surface. Thus, the S protein, which on itself was transported to the plasma membrane, was retained in the Golgi complex by its association with the M protein. Because coronaviruses bud at pre-Golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. Yet, the self-association of the MHV envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly.

scholarly journals Enhanced Virulence Mediated by the Murine Coronavirus, Mouse Hepatitis Virus Strain JHM, Is Associated with a Glycine at Residue 310 of the Spike Glycoprotein

2003 ◽  
Vol 77 (19) ◽  
pp. 10260-10269 ◽  
Author(s):  
Evelena Ontiveros ◽  
Taeg S. Kim ◽  
Thomas M. Gallagher ◽  
Stanley Perlman
Keyword(s):  
Virus Strain ◽  
Hepatitis Virus ◽  
Neurological Diseases ◽  
Mouse Hepatitis Virus ◽  
Neutralizing Antibody ◽  
Lateral Spread ◽  
S Protein ◽  
Acute Encephalitis ◽  
The Central Nervous System

ABSTRACT The coronavirus, mouse hepatitis virus strain JHM, causes acute and chronic neurological diseases in rodents. Here we demonstrate that two closely related virus variants, both of which cause acute encephalitis in susceptible strains of mice, cause markedly different diseases if mice are protected with a suboptimal amount of an anti-JHM neutralizing antibody. One strain, JHM.SD, caused acute encephalitis, while infection with JHM.IA resulted in no acute disease. Using recombinant virus technology, we found that the differences between the two viruses mapped to the spike (S) glycoprotein and that the two S proteins differed at four amino acids. By engineering viruses that differed by only one amino acid, we identified a serine-to-glycine change at position 310 of the S protein (S310G) that recapitulated the more neurovirulent phenotype. The increased neurovirulence mediated by the virus encoding glycine at position S310 was not associated with a different tropism within the central nervous system (CNS) but was associated with increased lateral spread in the CNS, leading to significantly higher brain viral titers. In vitro studies revealed that S310G was associated with decreased S1-S2 stability and with enhanced ability to mediate infection of cells lacking the primary receptor for JHM (“receptor-independent spread”). These enhanced fusogenic properties of viruses encoding a glycine at position 310 of the S protein may contribute to spread within the CNS, a tissue in which expression of conventional JHM receptors is low.

scholarly journals Soluble Receptor Potentiates Receptor-Independent Infection by Murine Coronavirus

2002 ◽  
Vol 76 (3) ◽  
pp. 950-958 ◽  
Author(s):  
Fumihiro Taguchi ◽  
Shutoku Matsuyama
Keyword(s):  
Monoclonal Antibody ◽  
Hepatitis Virus ◽  
Active Form ◽  
Mouse Hepatitis Virus ◽  
Soluble Receptor ◽  
Wild Type ◽  
S Protein ◽  
Bhk Cells ◽  
Direct Binding ◽  
Mediate Cell

ABSTRACT Mouse hepatitis virus (MHV) infection spreads from MHV-infected DBT cells, which express the MHV receptor CEACAM1 (MHVR), to BHK cells, which are devoid of the receptor, by intercellular membrane fusion (MHVR-independent fusion). This mode of infection is a property of wild-type (wt) JHMV cl-2 virus but is not seen in cultures infected with the mutant virus JHMV srr7. In this study, we show that soluble MHVR (soMHVR) potentiates MHVR-independent fusion in JHMV srr7-infected cultures. Thus, in the presence of soMHVR, JHMV srr7-infected DBT cells overlaid onto BHK cells induce BHK cell syncytia and the spread of JHMV srr7 infection. This does not occur in the absence of soMHVR. soMHVR also enhanced wt virus MHVR-independent fusion. These effects were dependent on the concentration of soMHVR in the culture and were specifically blocked by the anti-MHVR monoclonal antibody CC1. Together with these observations, direct binding of soMHVR to the virus spike (S) glycoprotein as revealed by coimmunoprecipitation demonstrated that the effect is mediated by the binding of soMHVR to the S protein. Furthermore, fusion of BHK cells expressing the JHMV srr7 S protein was also induced by soMHVR. These results indicated that the binding of soMHVR to the S protein expressed on the DBT cell surface potentiates the fusion of MHV-infected DBT cells with nonpermissive BHK cells. We conclude that the binding of soMHVR to the S protein converts the S protein to a fusion-active form competent to mediate cell-cell fusion, in a fashion similar to the fusion of virus and cell membranes.

scholarly journals Requirements for the Assembly and Release of Newcastle Disease Virus-Like Particles

2006 ◽  
Vol 80 (22) ◽  
pp. 11062-11073 ◽  
Author(s):  
Homer D. Pantua ◽  
Lori W. McGinnes ◽  
Mark E. Peeples ◽  
Trudy G. Morrison
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Plasma Membranes ◽  
Matrix Protein ◽  
Viral Proteins ◽  
M Protein ◽  
Virion Assembly ◽  
Virus Like Particles ◽  
Necessary And Sufficient

ABSTRACT Paramyxoviruses, such as Newcastle disease virus (NDV), assemble in and bud from plasma membranes of infected cells. To explore the role of each of the NDV structural proteins in virion assembly and release, virus-like particles (VLPs) released from avian cells expressing all possible combinations of the nucleoprotein (NP), membrane or matrix protein (M), an uncleaved fusion protein (F-K115Q), and hemagglutinin-neuraminidase (HN) protein were characterized for densities, protein content, and efficiencies of release. Coexpression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3 and 83.8% ± 1.1%, respectively) similar to those of authentic virions. Expression of M protein alone, but not NP, F-K115Q, or HN protein individually, resulted in efficient VLP release, and expression of all different combinations of proteins in the absence of M protein did not result in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F, and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by coimmunoprecipitation. The colocalization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M-HN and M-NP interactions are responsible for incorporation of HN and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein.

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 Putative cis-Acting Stem-Loops in the 5′ Untranslated Region of the Severe Acute Respiratory Syndrome Coronavirus Can Substitute for Their Mouse Hepatitis Virus Counterparts

2006 ◽  
Vol 80 (21) ◽  
pp. 10600-10614 ◽  
Author(s):  
Hyojeung Kang ◽  
Min Feng ◽  
Megan E. Schroeder ◽  
David P. Giedroc ◽  
Julian L. Leibowitz
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Regulatory Sequence ◽  
Stem Loop ◽  
Cis Acting ◽  
Transcriptional Regulatory ◽  
Similar Phenotype ◽  
Transcriptional Regulatory Sequence

ABSTRACT Consensus covariation-based secondary structural models for the 5′ 140 nucleotides of the 5′ untranslated regions (5′UTRs) from mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SCoV) were developed and predicted three major helical stem-loop structures, designated stem-loop 1 (SL1), SL2, and SL4. The SCoV 5′UTR was predicted to contain a fourth stem-loop, named SL3, in which the leader transcriptional regulatory sequence (TRS) is folded into a hairpin loop. cDNAs corresponding to MHV/SCoV chimeric genomes were constructed by replacing the complete MHV 5′UTR with the corresponding SCoV sequence and by separately replacing MHV 5′UTR putative SL1, putative SL2, TRS, and putative SL4 with the corresponding SCoV sequences. Chimeric genomes were transcribed in vitro, and viruses were recovered after electroporation into permissive cells. Genomes in which the MHV 5′UTR SL1, SL2, and SL4 were individually replaced by their SCoV counterparts were viable. Chimeras containing the complete SCoV 5′UTR or the predicted SCoV SL3 were not viable. A chimera containing the SCoV 5′UTR in which the SCoV TRS was replaced with the MHV TRS was also not viable. The chimera containing the entire SCoV 5′UTR failed to direct the synthesis of any virus-specific RNA. Replacing the SCoV TRS with the MHV TRS in the MHV/5′UTR SCoV chimera permitted the synthesis of minus-sense genome-sized RNA but did not support the production of positive- or minus-sense subgenomic RNA7. A similar phenotype was obtained with the MHV/SCoV SL3 chimera. These results suggest a role for the TRS in the replication of minus-sense genomic RNA in addition to its known function in subgenomic RNA synthesis.

Sign in / Sign up

Export Citation Format

Share Document