scholarly journals Baculovirus GP64 Disulfide Bonds: the Intermolecular Disulfide Bond of Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Is Not Essential for Membrane Fusion and Virion Budding

2010 ◽  
Vol 84 (17) ◽  
pp. 8584-8595 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Low Ph ◽  
Autographa Californica ◽  
Viral Fusion Protein ◽  
Intermolecular Disulfide Bond

ABSTRACT The GP64 envelope glycoprotein of the Autographa californica nucleopolyhedrovirus (AcMNPV) is a class III viral membrane fusion protein that is triggered by low pH during entry. Unlike most other viral fusion protein trimers, the monomers of GP64 are covalently linked to each other within the trimer by a single intermolecular disulfide bond (Cys24—Cys372). Single or paired alanine substitutions for Cys24 and Cys372 resulted in lower-efficiency transport of GP64 to the cell surface. Surprisingly, these mutated GP64s induced syncytium formation, and normalized fusion activities were approximately 30% of that from wild-type (WT) GP64. Heat treatment (37°C) did not further reduce fusion activity of GP64 constructs with a disrupted intermolecular disulfide bond, suggesting that the GP64 trimers were relatively thermostable in the absence of the intermolecular disulfide bond. In addition, analysis of binding by a conformation-specific monoclonal antibody (MAb) suggested that the low-pH-induced refolding of those GP64 constructs was generally similar to that of WT GP64. In addition to its critical role in membrane fusion, GP64 is also necessary for efficient budding. When GP64 constructs containing a disrupted intermolecular disulfide bond (Cys24—Cys372) were displayed at the cell surface at levels comparable to those of WT GP64, virion budding efficiency ranged from approximately 39 to 88%, indicating that the intermolecular disulfide bond is not required for virion budding. However, GP64 proteins with a disrupted intermolecular disulfide could not rescue a GP64-null bacmid. We also examined the 6 conserved intramolecular disulfide bonds using single and paired alanine substitution mutations. None of the GP64 constructs with disrupted intramolecular disulfide bonds were capable of mediating pH-triggered membrane fusion, indicating that the intramolecular disulfide bonds are all necessary for membrane fusion. Thus, while the intramolecular disulfide bonds of GP64 appear to serve critical roles in membrane fusion, the unusual intermolecular disulfide bond was not critical for membrane fusion or virion budding yet appears to play an unknown role in viral infectivity.

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 A Functional F Analogue of Autographa californica Nucleopolyhedrovirus GP64 from the Agrotis segetum Granulovirus

2008 ◽  
Vol 82 (17) ◽  
pp. 8922-8926 ◽  
Author(s):  
Feifei Yin ◽  
Manli Wang ◽  
Ying Tan ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Plutella Xylostella ◽  
Syncytium Formation ◽  
Low Ph ◽  
Autographa Californica ◽  
Agrotis Segetum ◽  
Induced Membrane ◽  
F Protein ◽  
Autographa Californica Nucleopolyhedrovirus

ABSTRACT The envelope fusion protein F of Plutella xylostella granulovirus is a computational analogue of the GP64 envelope fusion protein of Autographa californica nucleopolyhedrovirus (AcMNPV). Granulovirus (GV) F proteins were thought to be unable to functionally replace GP64 in the AcMNPV pseudotyping system. In the present study the F protein of Agrotis segetum GV (AgseGV) was identified experimentally as the first functional GP64 analogue from GVs. AgseF can rescue virion propagation and infectivity of gp64-null AcMNPV. The AgseF-pseudotyped AcMNPV also induced syncytium formation as a consequence of low-pH-induced membrane fusion.

scholarly journals Role of Metastability and Acidic pH in Membrane Fusion by Tick-Borne Encephalitis Virus

2001 ◽  
Vol 75 (16) ◽  
pp. 7392-7398 ◽  
Author(s):  
Karin Stiasny ◽  
Steven L. Allison ◽  
Christian W. Mandl ◽  
Franz X. Heinz
Keyword(s):  
Influenza Virus ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Low Ph ◽  
Class I ◽  
Acidic Ph ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Tick Borne Encephalitis ◽  
Tbe Virus

ABSTRACT The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.

scholarly journals Functional Analysis of the Transmembrane (TM) Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein: Substitution of Heterologous TM Domains

2008 ◽  
Vol 82 (7) ◽  
pp. 3329-3341 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Low Ph ◽  
Autographa Californica ◽  
Virus Infectivity ◽  
Type I ◽  
Gpi Anchor ◽  
Tm Domain ◽  
Hiv 1

ABSTRACT GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23-amino-acid GP64 TM domain with corresponding TM domain sequences from a range of viral and cellular type I membrane proteins, including Orgyia pseudotsugata MNPV (OpMNPV) GP64 and F, thogotovirus GP75, Lymantria dispar MNPV (LdMNPV) F, human immunodeficiency virus type 1 (HIV-1) GP41, human CD4 and glycophorin A (GpA), and influenza virus hemagglutinin (HA), and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments with Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low-pH treatment, but only those containing TM domains from CD4, GpA, OpMNPV GP64, and thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64 null virus, whereas those with TM domains from OpMNPV GP64 and thogotovirus GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were replaced only by TM domains from related viral membrane proteins.

scholarly journals Membrane Interactions of the Tick-Borne Encephalitis Virus Fusion Protein E at Low pH

2002 ◽  
Vol 76 (8) ◽  
pp. 3784-3790 ◽  
Author(s):  
Karin Stiasny ◽  
Steven L. Allison ◽  
Juliane Schalich ◽  
Franz X. Heinz
Keyword(s):  
Fusion Protein ◽  
Membrane Binding ◽  
Low Ph ◽  
Encephalitis Virus ◽  
Soluble Form ◽  
Viral Fusion ◽  
Sequence Element ◽  
Viral Fusion Protein ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

ABSTRACT Membrane fusion of the flavivirus tick-borne encephalitis virus is triggered by the mildly acidic pH of the endosome and is mediated by envelope protein E, a class II viral fusion protein. The low-pH trigger induces an oligomeric rearrangement in which the subunits of the native E homodimers dissociate and the monomeric subunits then reassociate into homotrimers. Here we provide evidence that membrane binding is mediated by the intermediate monomeric form of E, generated by low-pH-induced dissociation of the dimer. Liposome coflotation experiments revealed that association with target membranes occurred only when liposomes were present at the time of acidification, whereas pretreating virions at low pH in the absence of membranes resulted in the loss of their ability to stably attach to liposomes. With the cleavable cross-linker ethylene glycolbis(succinimidylsuccinate), it was shown that a truncated soluble form of the E protein (sE) could bind to membranes only when the dimers were free to dissociate at low pH, and binding could be blocked by a monoclonal antibody that recognizes the fusion peptide, which is at the distal tip of the E monomer but is buried in the native dimer. Surprisingly, analysis of the membrane-associated sE proteins revealed that they had formed trimers. This was unexpected because this protein lacks a sequence element in the C-terminal stem-anchor region, which was shown to be essential for trimerization in the absence of a target membrane. It can therefore be concluded that the formation of a trimeric form of sE is facilitated by membrane binding. Its stability is apparently maintained by contacts between the ectodomains only and is not dependent on sequence elements in the stem-anchor region as previously assumed.  

scholarly journals Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH

2006 ◽  
Vol 80 (22) ◽  
pp. 10931-10941 ◽  
Author(s):  
Rachel M. Schowalter ◽  
Stacy E. Smith ◽  
Rebecca Ellis Dutch
Keyword(s):  
Fusion Protein ◽  
Cleavage Site ◽  
Low Ph ◽  
Human Metapneumovirus ◽  
Proteolytic Processing ◽  
Luciferase Reporter ◽  
Transfected Cells ◽  
F Protein ◽  
Viral Fusion Protein ◽  
Glycosylation Sites

ABSTRACT Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.

scholarly journals Membrane Fusion Mediated by Baculovirus gp64 Involves Assembly of Stable gp64 Trimers into Multiprotein Aggregates

1998 ◽  
Vol 143 (5) ◽  
pp. 1155-1166 ◽  
Author(s):  
Ingrid Markovic ◽  
Helena Pulyaeva ◽  
Alexander Sokoloff ◽  
Leonid V. Chernomordik
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Structural Unit ◽  
Low Ph ◽  
Cross Linking ◽  
Target Cells ◽  
Sf9 Cells ◽  
Density Gradient Ultracentrifugation ◽  
Sds Page ◽  
Experimental Approaches

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell–surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell–cell contacts, suggests that such multimeric complexes represent a fusion machine.

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 Fusion Induced by a Class II Viral Fusion Protein, Semliki Forest Virus E1, Is Dependent on the Voltage of the Target Cell

2007 ◽  
Vol 81 (20) ◽  
pp. 11218-11225 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Margaret Kielian ◽  
Fredric S. Cohen
Keyword(s):  
Fusion Protein ◽  
Target Cell ◽  
Semliki Forest Virus ◽  
Low Ph ◽  
Negative Potential ◽  
Class I ◽  
Viral Fusion ◽  
Positive Potential ◽  
Viral Fusion Protein ◽  
Voltage Polarity

ABSTRACT Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.

scholarly journals Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (10) ◽  
pp. 6216-6226 ◽  
Author(s):  
Sandra Dawe ◽  
Jennifer A. Corcoran ◽  
Eileen K. Clancy ◽  
Jayme Salsman ◽  
Roy Duncan
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Transmembrane Protein ◽  
Topological Analysis ◽  
Viral Fusion ◽  
Structural Motifs ◽  
Membrane Fusion Protein ◽  
Viral Fusion Protein ◽  
Fast Proteins ◽  
Tm Domain

ABSTRACT Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic Nexoplasmic/Ccytoplasmic topology. This topology results in the translocation of the smallest ectodomain (∼20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.

Sign in / Sign up

Export Citation Format

Share Document