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 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 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.

A Spike Protein-Dependent Cellular Factor Other Than the Viral Receptor Is Required for Mouse Hepatitis Virus Entry

Virology ◽  
1993 ◽  
Vol 196 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Kyoko Yokomori ◽  
Miyuki Asanaka ◽  
Stephen A. Stohlman ◽  
Michael M.C. Lai
Keyword(s):  
Hepatitis Virus ◽  
Virus Entry ◽  
Mouse Hepatitis Virus ◽  
Spike Protein ◽  
Viral Receptor ◽  
Cellular Factor

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

1998 ◽  
Vol 72 (2) ◽  
pp. 1606-1609 ◽  
Author(s):  
Susan T. Hingley ◽  
Isabelle Leparc-Goffart ◽  
Susan R. Weiss
Keyword(s):  
Cell Fusion ◽  
Glial Cells ◽  
Virus Strain ◽  
Hepatitis Virus ◽  
Acute Infection ◽  
Mouse Hepatitis Virus ◽  
Cell Types ◽  
Spike Protein ◽  
S Protein

ABSTRACT 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.

scholarly journals Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

2015 ◽  
Vol 89 (17) ◽  
pp. 8816-8827 ◽  
Author(s):  
Zhaohui Qian ◽  
Xiuyuan Ou ◽  
Luiz Gustavo Bentim Góes ◽  
Christina Osborne ◽  
Anna Castano ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein ◽  
S Protein ◽  
Receptor Proteins ◽  
Group A

ABSTRACTCoronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human β-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.IMPORTANCEMouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory β-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two β-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

scholarly journals Variations in Disparate Regions of the Murine Coronavirus Spike Protein Impact the Initiation of Membrane Fusion

2001 ◽  
Vol 75 (6) ◽  
pp. 2792-2802 ◽  
Author(s):  
Dawn K. Krueger ◽  
Sean M. Kelly ◽  
Daniel N. Lewicki ◽  
Rosanna Ruffolo ◽  
Thomas M. Gallagher
Keyword(s):  
Tissue Culture ◽  
Membrane Fusion ◽  
Cultured Cells ◽  
Hepatitis Virus ◽  
Fusion Reaction ◽  
Spike Protein ◽  
Soluble Form ◽  
Fusion Activity ◽  
Murine Hepatitis Virus

ABSTRACT The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.

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