scholarly journals HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion

2011 ◽  
Vol 40 (4) ◽  
pp. 489-502 ◽  
Author(s):  
Kelly Sackett ◽  
Allan TerBush ◽  
David P. Weliky
Keyword(s):  
Protein Aggregation ◽  
Host Cell ◽  
Cell Fusion ◽  
Ph Dependence ◽  
Membrane Binding ◽  
Vesicle Fusion ◽  
Helix Bundle ◽  
Hiv Gp41

scholarly journals HIV Cell Fusion Assay

2013 ◽  
Vol 19 (1) ◽  
pp. 108-118 ◽  
Author(s):  
Elizabeth B. Smith ◽  
Robert A. Ogert ◽  
David Pechter ◽  
Artjohn Villafania ◽  
Susan J. Abbondanzo ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Chemokine Receptor ◽  
Viral Entry ◽  
Envelope Glycoprotein ◽  
Laser Scanning ◽  
Viral Envelope ◽  
Fusion Event ◽  
Cxcr4 Signaling ◽  
Hiv Entry

The health and disease-related biology of the CXCR4 chemokine receptor presents the challenge of finding a small molecule that can bind CXCR4 and block T-cell tropic human immunodeficiency virus type 1 (HIV-1) cell entry, while preserving the ability of CXCR4 to respond to its native ligand, CXCL12. HIV entry into the host cell involves the interaction of the viral envelope glycoprotein gp120 binding to CD4, followed by a rearrangement in gp120, and subsequent interaction with the chemokine receptor CXCR4 or CCR5. These initial events can be re-created in a cell fusion assay that represents a surrogate system, mimicking the early stages of viral entry via these host cell receptors. In the current study, a T-tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from the T-tropic HIV NL4-3 and HeLa cells expressing CD4 and CXCR4. Detection of the cell fusion event was based on a Gal4/VP16-activated β-lactamase signal and was measured by automated microscopy or laser scanning plate cytometry. Changes in morphology associated with cell fusion were combined with β-lactamase activity to generate results with robust assay statistics in both 384-well and 1536-well plates. Compounds were subsequently characterized by CXCR4 signaling assays to eliminate functional antagonists and allow the identification of a function-sparing HIV entry inhibitor.

scholarly journals Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

2020 ◽  
Author(s):  
Hanh T. Nguyen ◽  
Shijian Zhang ◽  
Qian Wang ◽  
Saumya Anang ◽  
Jia Wang ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Cleavage Site ◽  
Virus Entry ◽  
Cytoplasmic Tail ◽  
Syncytium Formation ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
Cytopathic Effects ◽  
Furin Cleavage Site

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

scholarly journals Protein aggregation in C-phycocyanin. Studies at very low concentrations with the photoelectric scanner of the ultracentrifuge

1971 ◽  
Vol 122 (4) ◽  
pp. 421-426 ◽  
Author(s):  
R. MacColl ◽  
J. J. Lee ◽  
D. S. Berns
Keyword(s):  
Protein Aggregation ◽  
Temperature Dependence ◽  
Equilibrium Constant ◽  
Ph Dependence ◽  
Sedimentation Velocity ◽  
Scanning System ◽  
High Concentration ◽  
Low Concentrations ◽  
Increasing Temperature ◽  
Schlieren Optics

Solutions of C-phycocyanin of very low concentrations were examined by sedimentation-velocity studies in the Spinco model E ultracentrifuge equipped with a photoelectric scanning system and a monochromator. At sufficiently low concentrations complete disaggregation from the hexamer to the monomer was observed. The equilibrium constant of monomer to hexamer was estimated to be approx. 1030. For studies of aggregation over the complete range of concentration, C-phycocyanins from Phormidium luridum and Lyngbya sp. were used. Sedimentation-velocity studies at high concentration with schlieren optics are reported for C-phycocyanins from Anabaena variabilis and Lyngbya sp. The pH-dependence of aggregation and the temperature-dependence of trimer–hexamer equilibrium for phycocyanins from these algae were found to be similar to those of other C-phycocyanins. The principal feature of the pH-dependence is the dominance of hexamers at the isoelectric point. Increasing temperature increased the amount of hexamer and decreased the amount of trimer.

The Hydrophobic Pocket Contributes to the Structural Stability of the N-Terminal Coiled Coil of HIV gp41 but Is Not Required for Six-Helix Bundle Formation

Biochemistry ◽  
2003 ◽  
Vol 42 (17) ◽  
pp. 4945-4953 ◽  
Author(s):  
John J. Dwyer ◽  
Aisha Hasan ◽  
Karen L. Wilson ◽  
Jonathan M. White ◽  
Thomas J. Matthews ◽  
...  
Keyword(s):  
Structural Stability ◽  
Coiled Coil ◽  
Hydrophobic Pocket ◽  
Helix Bundle ◽  
Hiv Gp41

scholarly journals Induction of Syncytia by Neuropathogenic Murine Leukemia Viruses Depends on Receptor Density, Host Cell Determinants, and the Intrinsic Fusion Potential of Envelope Protein

1999 ◽  
Vol 73 (11) ◽  
pp. 9377-9385 ◽  
Author(s):  
Maeran Chung ◽  
Krishnakumar Kizhatil ◽  
Lorraine M. Albritton ◽  
Glen N. Gaulton
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Syncytium Formation ◽  
Receptor Expression ◽  
Receptor Density ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Murine Leukemia ◽  
Nih 3T3 Cells

ABSTRACT Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.

scholarly journals Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein

1999 ◽  
Vol 73 (6) ◽  
pp. 5034-5042 ◽  
Author(s):  
Tatiana Zavorotinskaya ◽  
Lorraine M. Albritton
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Surface Protein ◽  
Host Cells ◽  
Virus Surface ◽  
Fusion Defect ◽  
Murine Leukemia ◽  
Cell Cell

ABSTRACT Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

2020 ◽  
Author(s):  
Hanh T. Nguyen ◽  
Shijian Zhang ◽  
Qian Wang ◽  
Saumya Anang ◽  
Jia Wang ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Cleavage Site ◽  
Virus Entry ◽  
Cytoplasmic Tail ◽  
Syncytium Formation ◽  
Spike Glycoprotein ◽  
Furin Cleavage ◽  
Cytopathic Effects ◽  
Furin Cleavage Site

ABSTRACTSARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent.IMPORTANCEThe development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.

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