scholarly journals Reevaluating Herpes Simplex Virus Hemifusion

2010 ◽  
Vol 84 (22) ◽  
pp. 11814-11821 ◽  
Author(s):  
Julia O. Jackson ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Viral Fusion Proteins ◽  
Simplex Virus

ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

scholarly journals Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

Virology ◽  
2014 ◽  
Vol 460-461 ◽  
pp. 128-137 ◽  
Author(s):  
Martina Maric ◽  
Alison C. Haugo ◽  
William Dauer ◽  
David Johnson ◽  
Richard J. Roller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Envelope ◽  
Herpes Simplex Virus Type ◽  
Fusion Proteins ◽  
Viral Fusion ◽  
Nuclear Envelope Breakdown ◽  
Viral Fusion Proteins ◽  
Simplex Virus

scholarly journals A New Class of Receptor for Herpes Simplex Virus Has Heptad Repeat Motifs That Are Common to Membrane Fusion Proteins

2005 ◽  
Vol 79 (12) ◽  
pp. 7419-7430 ◽  
Author(s):  
Aleida Perez ◽  
Qing-Xue Li ◽  
Pilar Perez-Romero ◽  
Gregory DeLassus ◽  
Santiago R. Lopez ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Surface Membrane ◽  
Heptad Repeat ◽  
Expression Cloning ◽  
Viral Fusion ◽  
Α Helix ◽  
Simplex Virus

ABSTRACT We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated hfl-B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an α-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat α-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion.

scholarly journals Identification of an N-Terminal Trimeric Coiled-Coil Core within Arenavirus Glycoprotein 2 Permits Assignment to Class I Viral Fusion Proteins

2006 ◽  
Vol 80 (12) ◽  
pp. 5897-5907 ◽  
Author(s):  
Bruno Eschli ◽  
Katharina Quirin ◽  
Alexander Wepf ◽  
Jacqueline Weber ◽  
Rolf Zinkernagel ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Gel Filtration ◽  
Class I ◽  
Spherical Particles ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Helical Peptide ◽  
Viral Fusion Proteins ◽  
Α Helix

ABSTRACT The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal α-helix. An α-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second α-helical peptide corresponding to a predicted C-terminal α-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of ∼230 and ∼440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.

scholarly journals A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

1999 ◽  
Vol 10 (12) ◽  
pp. 4191-4200 ◽  
Author(s):  
David H. Kingsley ◽  
Ali Behbahani ◽  
Afshin Rashtian ◽  
Gary W. Blissard ◽  
Joshua Zimmerberg
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Fusion Proteins ◽  
Critical Role ◽  
Low Ph ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Heptad Repeats

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

scholarly journals Structure-Based Mutations in the Herpes Simplex Virus 1 Glycoprotein B Ectodomain Arm Impart a Slow-Entry Phenotype

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Qing Fan ◽  
Sarah J. Kopp ◽  
Sarah A. Connolly ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Bacterial Artificial Chromosome ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Elevated Temperatures ◽  
Artificial Chromosome ◽  
Glycoprotein B ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus

ABSTRACTGlycoprotein B (gB) is the conserved herpesvirus fusion protein, and it is required for the entry of herpesviruses. The structure of the postfusion conformation of gB has been solved for several herpesviruses; however, the gB prefusion crystal structure and the details of how the protein refolds from a prefusion to a postfusion form to mediate fusion have not been determined. Using structure-based mutagenesis, we previously reported that three mutations (I671A, H681A, and F683A) in the C-terminal arm of the gB ectodomain greatly reduced cell-cell fusion. This fusion deficit could be rescued by the addition of a hyperfusogenic mutation, suggesting that the gB triple mutant was not misfolded. Using a bacterial artificial chromosome (BAC), we constructed two independent herpes simplex virus 1 mutant strains (gB 3A) carrying the three arm mutations. The gB 3A viruses have 200-fold smaller plaques than the wild-type virus and demonstrate remarkably delayed entry into cells. Single-step and multistep growth curves show that gB 3A viruses have delayed replication kinetics. Interestingly, incubation at 40°C promoted the entry of the gB 3A viruses. We propose that the gB 3A viruses’ entry deficit is due to a loss of interactions between residues in the gB C-terminal arm and the coiled-coil core of gB. The results suggest that the triple alanine mutation may destabilize the postfusion gB conformation and/or stabilize the prefusion gB conformation and that exposure to elevated temperatures can overcome the defect in gB 3A viruses.IMPORTANCEBecause of its complexity, the mechanism of herpesvirus entry into cells is not well understood. Our study investigated one of the most important unanswered questions about herpesvirus entry; i.e., how does the herpesvirus fusion protein gB mediate membrane fusion? gB is an essential protein that is conserved in all herpesviruses and is thought to undergo a conformational change to provide the energy to fuse the viral and cellular membranes. Using our understanding of the structure of gB, we designed mutations in the gB “arm” region that we predicted would impede gB function. We introduced these mutations into herpes simplex virus 1 by using a bacterial artificial chromosome, and the mutant virus exhibited a drastically delayed rate of entry. This entry defect was rescued by incubation at elevated temperatures, supporting a hypothesis that the engineered mutations altered the energetics of gB refolding. This study supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and the execution of membrane fusion, thus providing key details about the function of gB in herpesvirus-mediated fusion and subsequent virus entry.

scholarly journals The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex

2003 ◽  
Vol 77 (16) ◽  
pp. 8801-8811 ◽  
Author(s):  
Berend Jan Bosch ◽  
Ruurd van der Zee ◽  
Cornelis A. M. de Haan ◽  
Peter J. M. Rottier
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Limited Proteolysis ◽  
Fusion Peptide ◽  
Spike Protein ◽  
Class I ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Viral Fusion Protein

ABSTRACT Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure ∼14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.

scholarly journals Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Imane El Kasmi ◽  
Bita Khadivjam ◽  
Miki Lackman ◽  
Johanne Duron ◽  
Eric Bonneil ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Viral Proteins ◽  
Life Cycles ◽  
Viral Fusion ◽  
Herpes Simplex Virus 1 ◽  
Synaptotagmin 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTEnveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are unusual, as they fuse with a number of cellular compartments throughout their life cycles. As uncontrolled fusion of the host membranes should be avoided in these events, tight regulation of the viral fusion machinery is critical. While studying herpes simplex virus 1 (HSV-1) glycoprotein gM, we identified the cellular protein E-Syt1 (extended synaptotagmin 1) as an interaction partner. The interaction took place in both infected and transfected cells, suggesting other viral proteins were not required for the interaction. Most interestingly, E-Syt1 is a member of the synaptotagmin family of membrane fusion regulators. However, the protein is known to promote the tethering of the endoplasmic reticulum (ER) to the plasma membrane. We now show that E-Syt1, along with the related E-Syt3, negatively modulates viral release into the extracellular milieu, cell-to-cell viral spread, and viral entry, all processes that implicate membrane fusion events. Similarly, these E-Syt proteins impacted the formation of virus-induced syncytia. Altogether, these findings hint at the modulation of the viral fusion machinery by the E-Syt family of proteins.IMPORTANCEViruses typically encode their own fusion apparatus to enable them to enter cells. For many viruses, this means a single fusogenic protein. However, herpesviruses are large entities that express several accessory viral proteins to regulate their fusogenic activity. The present study hints at the additional participation of cellular proteins in this process, suggesting the host can also modulate viral fusion to some extent. Hence E-Syt proteins 1 and 3 seem to negatively modulate the different viral fusion events that take place during the HSV-1 life cycle. This could represent yet another innate immunity response to the virus.

scholarly journals Cascade of Events Governing Cell-Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gD, gH/gL, and gB

2010 ◽  
Vol 84 (23) ◽  
pp. 12292-12299 ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Gary H. Cohen ◽  
Roselyn J. Eisenberg
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Structural Level ◽  
Viral Fusion Protein ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT Herpesviruses minimally require the envelope proteins gB and gH/gL for virus entry and cell-cell fusion; herpes simplex virus (HSV) additionally requires the receptor-binding protein gD. Although gB is a class III fusion protein, gH/gL does not resemble any documented viral fusion protein at a structural level. Based on those data, we proposed that gH/gL does not function as a cofusogen with gB but instead regulates the fusogenic activity of gB. Here, we present data to support that hypothesis. First, receptor-positive B78H1-C10 cells expressing gH/gL fused with receptor-negative B78H1 cells expressing gB and gD (fusion in trans). Second, fusion occurred when gH/gL-expressing C10 cells preexposed to soluble gD were subsequently cocultured with gB-expressing B78 cells. In contrast, prior exposure of gB-expressing C10 cells to soluble gD did not promote subsequent fusion with gH/gL-expressing B78 cells. These data suggest that fusion involves activation of gH/gL by receptor-bound gD. Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. These data clearly show that gB acts as the HSV fusogen following activation by gD and gH/gL. We suggest the following steps leading to fusion: (i) conformational changes to gD upon receptor binding, (ii) alteration of gH/gL by receptor-activated gD, and (iii) upregulation of the fusogenic potential of gB following its interaction with activated gH/gL. The third step may be common to other herpesviruses.

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