scholarly journals Receptor-Independent Infection of Murine Coronavirus: Analysis by Spinoculation

2006 ◽  
Vol 80 (10) ◽  
pp. 4901-4908 ◽  
Author(s):  
Rie Watanabe ◽  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Cell Surface ◽  
Conformational Changes ◽  
Hepatitis Virus ◽  
Quantitative Estimation ◽  
Soluble Form ◽  
Activation Process ◽  
S Protein ◽  
Protein Activation ◽  
Naturally Occurring ◽  
Murine Coronavirus

ABSTRACT A highly neurovirulent murine coronavirus JHMV (wild-type [wt] JHMV) is known to spread from cells infected via the murine coronavirus mouse hepatitis virus receptor (MHVR) to cells without MHVR (MHVR-independent infection), whereas a mutant virus isolated from wt JHMV, srr7, spread only in an MHVR-dependent fashion. These observations were obtained by the overlay of JHMV-infected cells onto receptor-negative cells that are otherwise resistant to wt JHMV infection. MHVR-independent infection is hypothetically thought to be attributed to a naturally occurring fusion activation of the wt JHMV S protein, which did not occur in the case of srr7. Attachment of S protein on cells without MHVR during the S-protein activation process seems to be a key condition. Thus, in the present study, we tried to see whether wt JHMV virions that are attached on MHVR-negative cells are able to infect those cells. In order to make virions attach to the cell surface without MHVR, we have used spinoculation, namely, the centrifugation of cells together with inoculated virus at 3,000 rpm for 2 h. This procedure forces viruses to attach to the cell surface, as revealed by quantitative estimation of attached virions by real-time PCR and also facilitated wt JHMV infection to MHVR-negative cells, but failed to do so for srr7. Virions of both wt and srr7 attached on MHVR-negative cells by spinoculation were facilitated for infection in the presence of a soluble form of MHVR that induces conformational changes of both wt and srr7. It was further revealed that wt JHMV S1, but not srr7, was released from the cell surface when S protein was expressed on cells. These observations support the hypothesis that attachment of the virion to MHVR-negative cells is a critical step and that a unique feature of wt JHMV S1 to be released from S2 in a naturally occurring event is involved in an MHVR-independent infection.

scholarly journals Receptor-Induced Conformational Changes of Murine Coronavirus Spike Protein

2002 ◽  
Vol 76 (23) ◽  
pp. 11819-11826 ◽  
Author(s):  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Conformational Changes ◽  
Hepatitis Virus ◽  
Proteinase K ◽  
Soluble Form ◽  
Wild Type ◽  
S Protein ◽  
Direct Binding ◽  
Heptad Repeats ◽  
S Proteins ◽  
Murine Coronavirus

ABSTRACT Although murine coronavirus mouse hepatitis virus (MHV) enters cells by virus-cell membrane fusion triggered by its spike (S) protein, it is not well known how the S protein participates in fusion events. We reported that the soluble form of MHV receptor (soMHVR) transformed a nonfusogenic S protein into a fusogenic one (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In the present study, we demonstrate that soMHVR induces the conformational changes of the S protein, as shown by the proteinase digestion test. A cl-2 mutant, srr7, of the MHV JHM virus (JHMV) was digested with proteinase K after treatment with soMHVR, and the resultant S protein was analyzed by Western blotting using monoclonal antibody (MAb) 10G, specific for the membrane-anchored S2 subunit. A 58-kDa fragment, encompassing the two heptad repeats in S2, was detected when srr7 was digested after soMHVR treatment, while no band was seen when the virus was untreated. The appearance of the proteinase-resistant fragment was dependent on the temperature and time of srr7 incubation with soMHVR and also on the concentration of soMHVR. Coimmunoprecipitation indicated that the direct binding of soMHVR to srr7 S protein induced these conformational changes; this was also suggested by the inhibition of the changes following pretreatment of soMHVR with anti-MHVR MAb CC1. soMHVR induced conformational changes of the S proteins of wild-type (wt) JHMV cl-2, as well as revertants from srr7, srr7A and srr7B; however, a major proportion of these S proteins were resistant to proteinase K even without soMHVR treatment. The implications of this proteinase-resistant fraction are discussed. This is the first report on receptor-induced conformational changes of the membrane-anchored fragment of the coronavirus S protein.

scholarly journals N-Terminal Domain of the Murine Coronavirus Receptor CEACAM1 Is Responsible for Fusogenic Activation and Conformational Changes of the Spike Protein

2004 ◽  
Vol 78 (1) ◽  
pp. 216-223 ◽  
Author(s):  
Hideka S. Miura ◽  
Keiko Nakagaki ◽  
Fumihiro Taguchi
Keyword(s):  
Conformational Changes ◽  
Binding Assay ◽  
Solid Phase ◽  
Hepatitis Virus ◽  
Biological Activities ◽  
Protein S ◽  
Spike Protein ◽  
Soluble Form ◽  
S Protein ◽  
Solid Phase Binding

ABSTRACT The mouse hepatitis virus (MHV) receptor (MHVR), CEACAM1, has two different functions for MHV entry into cells: binding to MHV spike protein (S protein) and activation of the S protein to execute virus-cell membrane fusion, the latter of which is accompanied by conformational changes of the S protein. The MHVR comprising the N-terminal and fourth domains [R1(1,4)] displays these two activities, and the N domain is thought to be critical for binding to MHV. In this study, we have addressed whether or not the N domain alone is sufficient for these activities. We examined three types of soluble form MHVR (soMHVR), one consisting of the N domain alone [soR1(1)], one with the N and second domains [soR1(1,2)], and one [soR1(1,4)] expressed by recombinant baculoviruses. We assessed the abilities of these three types of soMHVR to bind to MHV, activate fusogenicity, and induce conformational changes of the S protein. All three types of soMHVR similarly bound to MHV, as examined by a solid-phase binding assay and neutralized MHV infectivity. They also activated S protein fusogenicity and induced its conformational changes with similar levels of efficiency. However, R1(1) expressed on the BHK cell surface failed to serve as a receptor in spite of a sufficient level of expression. The inability of expressed R1(1) to work as a receptor was due to the inaccessibility of virions to R1(1); however, these were accessible using the MHVR-specific monoclonal antibody CC1. These results collectively indicated that the N domain retains all biological activities necessary for receptor function.

scholarly journals Two-Step Conformational Changes in a Coronavirus Envelope Glycoprotein Mediated by Receptor Binding and Proteolysis

2009 ◽  
Vol 83 (21) ◽  
pp. 11133-11141 ◽  
Author(s):  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Hydrophobic Interactions ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Heptad Repeat ◽  
Soluble Form ◽  
S Protein

ABSTRACT The coronaviruses mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) utilize proteases to enter host cells. Upon receptor binding, the spike (S) proteins of both viruses are activated for membrane fusion by proteases, such as trypsin, present in the environment, facilitating virus entry from the cell surface. In contrast, in the absence of extracellular proteases, these viruses can enter cells via an endosomal pathway and utilize endosomal cathepsins for S protein activation. We demonstrate that the MHV-2 S protein uses multistep conformational changes for membrane fusion. After interaction with a soluble form of the MHV receptor (CEACAM1a), the metastable form of S protein is converted to a stable trimer, as revealed by mildly denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liposome-binding assays indicate that the receptor-bound virions are associated with the target membrane through hydrophobic interactions. The exposure of receptor-bound S protein to trypsin or cathepsin L (CPL) induces the formation of six-helix bundles (6HB), the final conformation. This trypsin- or CPL-mediated conversion to 6HB can be blocked by a heptad repeat peptide known to block the formation of 6HB. Although trypsin treatment enabled receptor-bound MHV-2 to enter from the cell surface, CPL failed to do so. Interestingly, consecutive treatment with CPL and then chlorpromazine enabled a portion of the virus to enter from cell surface. These results suggest that trypsin suffices for the induction of membrane fusion of receptor-primed S protein, but an additional unidentified cellular factor is required to trigger membrane fusion by CPL.

scholarly journals SARS-CoV-2–host cell surface interactions and potential antiviral therapies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aura-Bianca Butnariu ◽  
Alex Look ◽  
Marta Grillo ◽  
Tanveer A. Tabish ◽  
Michael J. McGarvey ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Conformational Changes ◽  
Surface Interactions ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Surface Receptors ◽  
Antiviral Therapies ◽  
The Us ◽  
Host Cell Surface

In this review, we reveal the latest developments at the interface between SARS-CoV-2 and the host cell surface. In particular, we evaluate the current and potential mechanisms of binding, fusion and the conformational changes of the spike (S) protein to host cell surface receptors, especially the human angiotensin-converting enzyme 2 (ACE2) receptor. For instance, upon the initial attachment, the receptor binding domain of the S protein forms primarily hydrogen bonds with the protease domain of ACE2 resulting in conformational changes within the secondary structure. These surface interactions are of paramount importance and have been therapeutically exploited for antiviral design, such as monoclonal antibodies. Additionally, we provide an insight into novel therapies that target viral non-structural proteins, such as viral RNA polymerase. An example of which is remdesivir which has now been approved for use in COVID-19 patients by the US Food and Drug Administration. Establishing further understanding of the molecular details at the cell surface will undoubtably aid the development of more efficacious and selectively targeted therapies to reduce the burden of COVID-19.

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 Conformational Changes in the Spike Glycoprotein of Murine Coronavirus Are Induced at 37°C either by Soluble Murine CEACAM1 Receptors or by pH 8

2003 ◽  
Vol 77 (2) ◽  
pp. 830-840 ◽  
Author(s):  
Bruce D. Zelus ◽  
Jeanne H. Schickli ◽  
Dianna M. Blau ◽  
Susan R. Weiss ◽  
Kathryn V. Holmes
Keyword(s):  
Conformational Change ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Virus Neutralization ◽  
Virus Infectivity ◽  
Soluble Receptor ◽  
Spike Glycoprotein ◽  
S Protein ◽  
Neutral Ph ◽  
Murine Coronavirus

ABSTRACT The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37°C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37°C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4°C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37°C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37°C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered SA59H716D virions with trypsin at 4°C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37°C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.

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 Protease-Mediated Entry via the Endosome of Human Coronavirus 229E

2008 ◽  
Vol 83 (2) ◽  
pp. 712-721 ◽  
Author(s):  
Miyuki Kawase ◽  
Kazuya Shirato ◽  
Shutoku Matsuyama ◽  
Fumihiro Taguchi
Keyword(s):  
Cell Surface ◽  
Protease Inhibitors ◽  
Hepatitis Virus ◽  
Cathepsin L ◽  
Syncytium Formation ◽  
Human Coronavirus ◽  
S Protein ◽  
Mode Of Entry ◽  
Group I ◽  
Human Coronavirus 229E

ABSTRACT Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry

2006 ◽  
Vol 1 (4) ◽  
pp. 1840-1851 ◽  
Author(s):  
Srinivasa-Gopalan Sampathkumar ◽  
Mark B Jones ◽  
Kevin J Yarema
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Quantitative Estimation ◽  
Sialic Acids
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