pH-Dependent Conformational Changes in the HCV NS3 Protein Modulate Its ATPase and Helicase Activities

The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Conformational changes of Newcastle disease virus envelope glycoproteins triggered by gangliosides.

We have investigated the conformational changes of Newcastle disease virus (NDV) glycoproteins in response to receptor binding, using 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) as a hydrophobicity-sensitive probe. Temperature- and pH-dependent conformational changes were detected in the presence of free bovine gangliosides. The fluorescence of bis-ANS was maximal at pH 5. The binding of bis-ANS to NDV was not affected by chemicals that denature the fusion glycoprotein, such as reducing agents, nor by the presence of neuraminidase inhibitors such as N-acetyl neuramicic acid. Gangliosides partially inhibited fusion and hemadsorption, but not neuraminidase hemagglutinin-neuraminidase glycoprotein (HN) activity. A conformational intermediate of HN, triggered by the presence of gangliosides acting as receptor mimics, was detected. Our results indicate that, upon binding to free gangliosides, HN undergoes a certain conformational change that does not affect the fusion glycoprotein.     

The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway.

The hemagglutinin of influenza virus (HA), an acid-activated membrane fusion protein, is synthesized in the endoplasmic reticulum and transported through the Golgi complex to the cell surface of infected cells as an uncleaved, fusion-incompetent precursor, HA0. The mature, proteolytically activated HA is known to undergo a rapid, irreversible, acid-induced conformational change which mediates membrane fusion and virus penetration. On the basis of antigenic modifications and the acquisition of trypsin susceptibility, we demonstrate here that HA0, while unable to cause fusion, is acid sensitive. It undergoes irreversible conformational changes quite similar to those of HA at mildly acidic pH (pH less than 6.0). The ectodomain of HA0 does not, however, acquire hydrophobic properties and the changes occur in a less concerted manner (the pH dependence is much broader and the rate of conversion slower). These differences are likely to account for the inability of acid-treated HA0 to trigger membrane fusion. It was shown, moreover, that HA0 acquired its acid-sensitive properties immediately following trimerization in the endoplasmic reticulum. Since HA0 did not convert to the acid form at any point during its intracellular transport, we concluded that the trans-Golgi compartment, known to be more acidic than the cytosol and involved in constitutive membrane transport, is not likely to have a pH less than 6.0.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp¹⁵³ and Glu¹⁵⁸, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His¹⁴⁸ was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

pH-dependent binding of the fluorophore bis-ANS to influenza virus reflects the conformational change of hemagglutinin

Binding of the fluorophore 1,1-bis(4-anili-no) naphthalene-5,5-disulfonic acid (bis-ANS) to influenza virus A/PR 8/34 is strongly enhanced at low pH. Binding is accompanied by a significant increase in fluorescence intensity. The binding and the fluorescence increase are associated with the low-pH induced conformational change of the viral spike protein, hemagglutinin, exposing hydrophobic binding sites. The data indicate that in addition to the hydrophobic N-terminus of HA2 other hydrophobic sequences of the HA ectodomain become accessible to bis-ANS at low pH. It is shown that the time course of the fluorescence increase of bis-ANS at low pH is determined by the conformational change of HA. The application of this assay for continuously monitoring the kinetics of the structural alteration in HA is discussed and its relevance for elucidating the temporal relationship between the conformational change of HA and virus-membrane fusion is outlined.Abbreviations HA hemagglutinin - BHA bromelain-solubilized ectodomain of HA - N-HA2 N-terminus of the HA2 subunit - PBS phosphate buffered saline - bis-ANS (1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid) - R18 octadecylrhodamine B chloride - FDQ fluorescence dequenching - RBC red blood cell Correspondence to: A. Herrmann     

Multistep regulation of membrane insertion of the fusion peptide of Semliki Forest virus

A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1(*)) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1(*) interaction with target membranes and trimerization. E1(*) was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.     

Membrane recognition by vesicular stomatitis virus involves enthalpy-driven protein-lipid interactions

Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.     

Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes

Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis-unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification.     

Pressure-induced fusogenic conformation of vesicular stomatitis virus glycoprotein

Vesicular stomatitis virus (VSV) is composed of a ribonucleoprotein core surrounded by a lipid envelope presenting an integral glycoprotein (G). The homotrimeric VSV G protein exhibits a membrane fusion activity that can be elicited by low pH. The fusion event is crucial to entry into the cell and disassembly followed by viral replication. To understand the conformational changes involved in this process, the effects of high hydrostatic pressure and urea on VSV particles and isolated G protein were investigated. With pressures up to 3.0 kbar VSV particles were converted into the fusogenic conformation, as measured by a fusion assay and by the binding of bis-ANS. The magnitude of the changes was similar to that promoted by lowering the pH. To further understand the relationship between stability and conversion into the fusion-active states, the stability of the G protein was tested against urea and high pressure. High urea produced a large red shift in the tryptophan fluorescence of G protein whereas pressure promoted a smaller change. Pressure induced equal fluorescence changes in isolated G protein and virions, indicating that virus inactivation induced by pressure is due to changes in the G protein. Fluorescence microscopy showed that pressurized particles were capable of fusing with the cell membrane without causing infection. We propose that pressure elicits a conformational change in the G protein, which maintains the fusion properties but suppresses the entry of the virus by endocytosis. Binding of bis-ANS indicates the presence of hydrophobic cavities in the G protein. Pressure also caused an increase in light scattering of VSV G protein, reinforcing the hypothesis that high pressure elicits the fusogenic activity of VSV G protein. This "fusion-intermediate state" induced by pressure has minor changes in secondary structure and is likely the cause of nonproductive infections.     

Membrane fusion induced by vesicular stomatitis virus depends on histidine protonation

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pH-induced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.     

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-induced alterations in the fusogenic spike protein of Semliki Forest virus

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.     

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.     

Reversible conformational changes and fusion activity of rabies virus glycoprotein.

In an attempt to understand the implication of the rabies virus glycoprotein (G) in the first steps of the viral cycle, we studied the pH dependence of virus-induced fusion and hemagglutination, as well as modifications of the structure and properties of the viral glycoprotein following pH acidification. Our results suggest that the G protein adopts at least three distinct configurations, each associated with different properties. At neutral pH, G did not fuse membranes or hemagglutinate erythrocytes. It was insensitive to digestion with bromelain and trypsin. At pH 6.4, the glycoprotein became sensitive to proteases. Hemagglutination was at its maximum and then sharply decreased with the pH. No fusion was detected. Aggregation of virus was also observed. The third configuration, at below pH 6.1, was associated with the appearance of fusion. Some neutralizing monoclonal antibodies were able to differentiate these three configurations. Preincubation of the virus at below pH 6 inhibited fusion, but this inhibition, like the structural modifications of the glycoprotein, was reversible when G was reincubated at neutral pH.     

Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH

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.     

pH-dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment of influenza virus hemagglutinin

A twenty amino acid hydrophobic peptide with the same sequence as that of the HA2 N-terminal segment of influenza virus hemagglutinin was synthesized and studied as to its fusion activity. The peptide caused rapid and efficient fusion of egg yolk phosphatidylcholine sonicated vesicles at acidic pH but not at neutral pH. The threshold pH was ca. 6.2 and the maximum fusion occurred at pH 4.8, the half-maximal pH for fusion being 5.6. The pH dependence was similar to that of the parent virus. The fusion efficiency was dependent on the ration of lipid to peptide, increasing with decreasing ratio. The fusion can be rapidly switched on and off by adjusting the pH, to the acidic side and neutral, respectively. The peptide with an acetylated or succinylated N-terminus also showed low pH-induced fusion activity but the pH range was shifted by ca. 1 unit to the acidic side. The results indicate that the HA2 hydrophobic segment in the virus fusion protein is directly involved in the fusion reaction and protonation of the acidic residues in the segment is required for the activity.     

A discrete stage of baculovirus GP64-mediated membrane fusion

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.     

Fusion of artificial lipid membranes induced by synthetic "fusion peptide" of arenaviruses

The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.