scholarly journals Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

2020 ◽  
Vol 6 (8) ◽  
pp. 2017-2022
Author(s):  
Victor K. Outlaw ◽  
Dale F. Kreitler ◽  
Debora Stelitano ◽  
Matteo Porotto ◽  
Anne Moscona ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Viral Fusion ◽  
Viral Fusion Protein

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 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 Identification and Characterization of the Putative Fusion Peptide of the Severe Acute Respiratory Syndrome-Associated Coronavirus Spike Protein

2005 ◽  
Vol 79 (11) ◽  
pp. 7195-7206 ◽  
Author(s):  
Bruno Sainz ◽  
Joshua M. Rausch ◽  
William R. Gallaher ◽  
Robert F. Garry ◽  
William C. Wimley
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Fusion Protein ◽  
Protein A ◽  
Fusion Peptide ◽  
Class I ◽  
Viral Fusion ◽  
Fusion Peptides ◽  
Viral Fusion Protein ◽  
Ww Ii ◽  
Heptad Repeats

ABSTRACT Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARSWW-I and SARSWW-II) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARSWW-I peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a β-sheet structure. Likewise, only SARSWW-I induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARSWW-I, we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.

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.

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