scholarly journals Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking

2007 ◽  
Vol 81 (21) ◽  
pp. 12019-12028 ◽  
Author(s):  
Hilde M. van der Schaar ◽  
Michael J. Rust ◽  
Barry-Lee Waarts ◽  
Heidi van der Ende-Metselaar ◽  
Richard J. Kuhn ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Dengue Virus ◽  
Membrane Fusion ◽  
High Ratio ◽  
Cell Entry ◽  
Viral Membrane ◽  
Virus Particles ◽  
Biochemical Assays ◽  
Particle Ratio ◽  
Infectious Unit

ABSTRACT In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.

scholarly journals How antibodies alter the cell entry pathway of dengue virus particles in macrophages

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nilda V. Ayala-Nunez ◽  
Tabitha E. Hoornweg ◽  
Denise P.I. van de Pol ◽  
Klaas A. Sjollema ◽  
Jacky Flipse ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Cell Entry ◽  
Virus Particles ◽  
Entry Pathway

scholarly journals COVID-19: The CaMKII_Like System of S Protein Drives Membrane Fusion and Induces Syncytial Multinucleated Giant Cells

2021 ◽  
Author(s):  
liu wenzhong ◽  
Li hualan
Keyword(s):  
Cell Membrane ◽  
Infected Cell ◽  
Membrane Fusion ◽  
Calcium Ions ◽  
Giant Cells ◽  
Multinucleated Giant Cells ◽  
S Protein ◽  
Viral Membrane ◽  
Ef Hand ◽  
Contact Position

COVID-19 is a unique disease characterized by extensive pulmonary thrombosis and infected syncytial multinucleated giant cells, relating to extensive tissue damage. The SARS-CoV-2 S protein on the membrane of infected cells can initiate receptor-dependent syncytia formation. To study the membrane fusion on S protein, we adopted structural domain search methods to analyze the structural and non-structural proteins of the SARS-COV-2 virus in this study. The results showed that the surface glycoprotein (S) had conserved domains of CaMKII: CaMKII_AD, CaATP_NAI, DUF4440, EF-hand, Protein kinase, and SnoaL-like. Comparing to SARS-COV and MERS, only the CaATP_NAI of SARS-COV-2 is in the contact position of the viral membrane and cell membrane. We believed that when the EF-hand domain (“YEQYIKWPWYIWLGF”) of S protein bound to calcium ions, S2 protein had CaMKII protein activities. After the S protein fusion peptide was inserted into the infected cell membrane and fixed the S2 protein on the cell membrane, the CaMKII_AD prompted the S2 protein to form HR1-HR2 six-helix bundles. The HR1-HR2 hexamer had three CaATP_NAI domains (“APAICHDGKAHFPRE”) near the viral membrane (contact position), the CaATPase activated by magnesium ions, and released energy through ATP phosphorylation. The CaATPase drove the HR1-HR2 hexamer fold irreversibly toward the viral membrane. Then the CaATP_NAI and CaMKII_AD domains extended to the outside and combined the viral membrane and the cell membrane so that the contact position formed a thin barrel structure. The hydrated calcium ions are gathered in the barrel structure to create a calcium bridge. The release action of water in contact position caused the instability of the double membrane, triggering lipid mixing and fusion of the membrane. CaATPases disassembled the barrel structure, and HR1-HR2 hexamer is fell into the cytoplasm. The viral membrane fused with the cell membrane on a large scale. The cytoplasmic contents of the virus mixed with the cell. The S protein of the infected cell may bind to the ACE2 receptor of another cell (or also an infected cell) and then achieved membrane fusion through a similar principle, forming cell syncytia, includes syncytial multinucleated giant cells. The membrane fusion could disrupt the calcium homeostasis in human body, and increased the risk of coagulation and vascular calcification.

scholarly journals Functional importance of dengue virus maturation: infectious properties of immature virions

2008 ◽  
Vol 89 (12) ◽  
pp. 3047-3051 ◽  
Author(s):  
Izabela A. Zybert ◽  
Heidi van der Ende-Metselaar ◽  
Jan Wilschut ◽  
Jolanda M. Smit
Keyword(s):  
Dengue Virus ◽  
Surface Protein ◽  
General Assumption ◽  
Wild Type Virus ◽  
Virus Maturation ◽  
Virus Particles ◽  
Lovo Cells ◽  
Particle Ratio ◽  
Infected Cells ◽  
Infectious Titre

Prior to the release of flavivirus particles from infected cells, the viral surface protein prM is cleaved to M by the cellular enzyme furin. For dengue virus (DENV), this maturation process appears to be very inefficient since a high proportion of progeny virions contain uncleaved prM. Furthermore, it has been reported that prM-containing DENV particles are infectious. These observations contradict the general assumption that prM processing is required to render virus particles infectious. Therefore, in this study, we reinvestigated the infectious properties of immature DENV virions. DENV particles were produced in furin-deficient LoVo cells. We observed that DENV-infected LoVo cells secrete high numbers of prM-containing particles. Subsequent analysis of the infectious titre revealed that immature particles lack the ability to infect cells, the infectious unit to particle ratio being 10 000-fold reduced compared with that of wild-type virus. Our results indicate that cleavage of prM to M is required for DENV infectivity.

scholarly journals Dynamics of SARS-CoV-2 Spike Proteins in Cell Entry: Control Elements in the Amino-Terminal Domains

mBio ◽  
2021 ◽  
Author(s):  
Enya Qing ◽  
Tom Kicmal ◽  
Binod Kumar ◽  
Grant M. Hawkins ◽  
Emily Timm ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Entry ◽  
Adaptive Changes ◽  
Amino Terminal ◽  
Spike Proteins ◽  
Terminal Domains

Adaptive changes that increase SARS-CoV-2 transmissibility may expand and prolong the coronavirus disease 2019 (COVID-19) pandemic. Transmission requires metastable and dynamic spike proteins that bind viruses to cells and catalyze virus-cell membrane fusion.

scholarly journals COVID-19: The CaMKII_Like System of S Protein Drives Membrane Fusion and Induces Syncytial Multinucleated Giant Cells

2021 ◽  
Author(s):  
liu wenzhong ◽  
Li hualan
Keyword(s):  
Cell Membrane ◽  
Infected Cell ◽  
Membrane Fusion ◽  
Calcium Ions ◽  
Giant Cells ◽  
Multinucleated Giant Cells ◽  
S Protein ◽  
Viral Membrane ◽  
Ef Hand ◽  
Contact Position

COVID-19 is a unique disease characterized by extensive pulmonary thrombosis and infected syncytial multinucleated giant cells, relating to extensive tissue damage. The SARS-CoV-2 S protein on the membrane of infected cells can initiate receptor-dependent syncytia formation. To study the membrane fusion on S protein, we adopted structural domain search methods to analyze the structural and non-structural proteins of the SARS-COV-2 virus in this study. The results showed that the surface glycoprotein (S) had conserved domains of CaMKII: CaMKII_AD, CaATP_NAI, DUF4440, EF-hand, Protein kinase, and SnoaL-like. Comparing to SARS-COV and MERS, only the CaATP_NAI of SARS-COV-2 is in the contact position of the viral membrane and cell membrane. We believed that when the EF-hand domain (“YEQYIKWPWYIWLGF”) of S protein bound to calcium ions, S2 protein had CaMKII protein activities. After the S protein fusion peptide was inserted into the infected cell membrane and fixed the S2 protein on the cell membrane, the CaMKII_AD prompted the S2 protein to form HR1-HR2 six-helix bundles. The HR1-HR2 hexamer had three CaATP_NAI domains (“APAICHDGKAHFPRE”) near the viral membrane (contact position), the CaATPase activated by magnesium ions, and released energy through ATP phosphorylation. The CaATPase drove the HR1-HR2 hexamer fold irreversibly toward the viral membrane. Then the CaATP_NAI and CaMKII_AD domains extended to the outside and combined the viral membrane and the cell membrane so that the contact position formed a thin barrel structure. The hydrated calcium ions are gathered in the barrel structure to create a calcium bridge. The release action of water in contact position caused the instability of the double membrane, triggering lipid mixing and fusion of the membrane. CaATPases disassembled the barrel structure, and HR1-HR2 hexamer is fell into the cytoplasm. The viral membrane fused with the cell membrane on a large scale. The cytoplasmic contents of the virus mixed with the cell. The S protein of the infected cell may bind to the ACE2 receptor of another cell (or also an infected cell) and then achieved membrane fusion through a similar principle, forming cell syncytia, includes syncytial multinucleated giant cells. The membrane fusion could disrupt the calcium homeostasis in human body, and increased the risk of coagulation and vascular calcification.

scholarly journals Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains

2015 ◽  
Vol 89 (11) ◽  
pp. 6093-6104 ◽  
Author(s):  
James T. Earnest ◽  
Michael P. Hantak ◽  
Jung-Eun Park ◽  
Tom Gallagher
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Target Cell ◽  
Influenza A ◽  
Virus Entry ◽  
Cell Entry ◽  
Membrane Microdomains ◽  
Enveloped Viruses ◽  
Viral Surface ◽  
Surface Glycoproteins

ABSTRACTCoronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases.IMPORTANCEEnveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus priming with greater precision. Implications of these findings extend to the use of virus entry antagonists, such as protease inhibitors, which might be most effective when localized to these microdomains.

scholarly journals A Hyperstabilizing Mutation in the Base of the Ebola Virus Glycoprotein Acts at Multiple Steps To Abrogate Viral Entry

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
J. Maximilian Fels ◽  
Jennifer S. Spence ◽  
Robert H. Bortz ◽  
Zachary A. Bornholdt ◽  
Kartik Chandran
Keyword(s):  
Membrane Fusion ◽  
Host Cell ◽  
Viral Entry ◽  
Ebola Virus ◽  
Cysteine Proteases ◽  
Cell Entry ◽  
Next Generation ◽  
Viral Membrane ◽  
Late Entry ◽  
Viral Membrane Fusion

ABSTRACTEbola virus (EBOV) causes highly lethal disease outbreaks against which no FDA-approved countermeasures are available. Although many host factors exploited by EBOV for cell entry have been identified, including host cell surface phosphatidylserine receptors, endosomal cysteine proteases, and the lysosomal cholesterol trafficking protein NPC1, key questions remain. Specifically, late entry steps culminating in viral membrane fusion remain enigmatic. Here, we investigated a set of glycoprotein (GP) mutants previously hypothesized to be entry defective and identified one mutation, R64A, that abolished infection with no apparent impact on GP expression, folding, or viral incorporation. R64A profoundly thermostabilized EBOV GP and rendered it highly resistant to proteolysisin vitro. Forward-genetics and cell entry studies strongly suggested that R64A’s effects on GP thermostability and proteolysis arrest viral entry at least at two distinct steps: the first upstream of NPC1 binding and the second at a late entry step downstream of fusion activation. Concordantly, toremifene, a small-molecule entry inhibitor previously shown to bind and destabilize GP, may selectively enhance the infectivity of viral particles bearing GP(R64A) at subinhibitory concentrations. R64A provides a valuable tool to further define the interplay between GP stability, proteolysis, and viral membrane fusion; to explore the rational design of stability-modulating antivirals; and to spur the development of next-generation Ebola virus vaccines with improved stability.IMPORTANCEEbola virus is a medically relevant virus responsible for outbreaks of severe disease in western and central Africa, with mortality rates reaching as high as 90%. Despite considerable effort, there are currently no FDA-approved therapeutics or targeted interventions available, highlighting the need of development in this area. Host-cell invasion represents an attractive target for antivirals, and several drug candidates have been identified; however, our limited understanding of the complex viral entry process challenges the development of such entry-targeting drugs. Here, we report on a glycoprotein mutation that abrogates viral entry and provides insights into the final steps of this process. In addition, the hyperstabilized phenotype of this mutant makes it useful as a tool in the discovery and design of stability-modulating antivirals and next-generation vaccines against Ebola virus.

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