Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion.

The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus (BV) and is involved in BV entry into the host cell by endocytosis. To determine whether gp64 alone was sufficient to mediate membrane fusion, the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 protein was transiently expressed in uninfected insect cells. Cells expressing the baculovirus gp64 protein were examined for membrane fusion activity by using a syncytium formation assay under various conditions of exposure to low pH. Cells expressing the gp64 protein mediated membrane fusion and syncytium formation in a pH-dependent manner. A pH of 5.5 or lower was required to induce membrane fusion. In addition, exposure of gp64-expressing cells to low pH for as little as 5 s was sufficient to induce gp64-mediated syncytium formation. These studies provide direct evidence that gp64 is a pH-dependent membrane fusion protein and suggest that gp64 is the protein responsible for fusion of the virion envelope with the endosome membrane during BV entry into the host cell by endocytosis.     

Low-pH-Dependent Fusion of Sindbis Virus with Receptor-Free Cholesterol- and Sphingolipid-Containing Liposomes

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.     

pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry.

Murine leukemia virus ecotropic and amphotropic envelope expression vectors were genetically engineered to generate truncations of the p15E TM cytoplasmic tail. The ecotropic construct CEET has the entire cytoplasmic tail of TM deleted, while the CEETR construct has only the R peptide portion of the tail deleted, thereby producing a TM subunit (p12E) that is identical to the one found in mature virions. The analogous amphotropic constructs were called CAET and CAETR. These envelopes, as opposed to their p15E TM counterparts, mediate cell-to-cell fusion at neutral pH in both transformed and nontransformed cell lines. Though the TM cytoplasmic domain is not required, its presence appears to augment such cell-to-cell fusion. This envelope-dependent fusion requires the presence of the viral receptor on the cell surface. Ecotropic virions bearing the p12E TM contain wild-type levels of the envelope complex and have near-normal titers. In contrast, virions which lack the cytoplasmic domain of TM (e.g., CEET) have 10- to 100-fold-lower titers but contain normal amounts of envelope. Both of the corresponding amphotropic virions contain normal amounts of envelope but have 10- to 100-fold-lower titers. Using immunofluorescent detection of envelope to monitor the fate of receptor-bound virions, we found that ecotropic murine leukemia virus envelope disappears from the cell surface while amphotropic envelope persists on the cell surface after virus binding. This pattern of immunofluorescence is consistent with the proposed routes of cell entry for these viruses, i.e., by endocytosis and direct fusion, respectively. In this assay, ecotropic virions bearing the genetically engineered p12E TM also appear to be internalized despite the ability of their envelope to mediate fusion at neutral pH in the same target cells. Our results show that direct fusion at neutral pH is a natural consequence of the surface expression of the mature ecotropic envelope and its receptor. We propose that the processing of the R peptide from the envelope TM (p15E) to yield p12E, at the time of virus budding or within virions, renders the envelope competent to fuse.     

Molecular mechanisms of virus spread and virion components as tools of virulence. A review

Despite of differences in replication strategy among virus families, some basic principles have remained similar. Analogous mechanisms govern virus entry into cells and the use of enzymes which direct the replication of the virus genome. The function of many cell surface receptors (such as glycosoaminoglycans, glycoproteins, proteins) which interact with viral capsid proteins or envelope glycoproteins has recently been elucidated. The list of cellular receptors (Table I) is still far from being final. The capsid components, similarly as the envelope glycoproteins, may form specific pocket like sites, which interact with the cell surface receptors. Neutralizing antibodies usually react with antigenic domains adjacent to the receptor binding site(s) and hamper the close contact inevitable for virion attachment. In the case of more complex viruses, such as herpes simplex virus, different viral glycoproteins interact with several cellular receptors. At progressed phase of adsorption the virions are engulfed into endocytic vesicles and the virion fusion domain(s) become(s) activated. The outer capsid components of reoviruses which participate in adsorption and fusion may get activated already in the lumen of digestive tract, i.e. before their engulfment by resorptive epithelium cells. Activation of the hydrophobic fusion domain(s) is a further important step allowing to pass through the lipid bilayer when penetrating the cell membrane in order to reach the cytosol. Activation of the virion fusion domain is accomplished by a conformation change, which occurs at acid pH (influenza virus hemagglutinin, sigma 1 protein of the reovirus particle) and/or after protease treatment. The herpes simplex virus fusion factors (gD and gH) undergo conformation changes by a pH-independent mechanism triggered due to interaction with the cell surface receptor(s) and mediated by mutual interactions with the viral envelope glycoproteins. The virion capsid or envelope components participating in the entry and membrane fusion are not the only tools of virulence. The correct function of virus coded proteins, which participate in replication of the viral genome, and/or in the supply of necessary nucleotides, may be very essential. In the case of enteroviruses, which RNA interacts with ribosomes directly, the correct configuration of the non-coding viral RNA sequence is crucial for initiation of translation occurring in the absence of the classical "cap" structure.     

Downregulation of Mcl-1 by daunorubicin pretreatment reverses resistance of breast cancer cells to TNF-related apoptosis-inducing ligand

The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from β-sheet conformation to an elongated coil and then spontaneously to an α-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.     

Characterization of Potent Fusion Inhibitors of Influenza Virus

New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes.     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

Functional analysis of hepatitis C virus envelope proteins, using a cell-cell fusion assay

Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.     

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.     

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.     

The role of target membrane sialic acid residues in the fusion activity of the influenza virus: the effect of two types of ganglioside on the kinetics of membrane merging

The influenza virus enters target cells via the action of hemagglutinin proteins (HA) inserted into the viral envelope. HA promotes membrane fusion between the viral envelope and endosomal membrane at low pH, following viral binding to sialic acid-containing receptors on target cells, and internalization by endocytosis. The effect of target membrane sialic acid residues on the fusion activity of the influenza virus towards model membranes was evaluated by both reduction, (i.e. treating somatic cells with neuraminidase- (NA-) prior to virus-cell interactions), and by supplementing liposomes with the gangliosides GD1a and GT1b. The harshness of the neuraminidase pretreatment of target cells required to affect virus-induced membrane merging was found to greatly depend on the assay conditions, i.e. whether a virus-cell prebinding step at neutral pH was included prior to acidification. Minor concentrations of neuraminidase were found to greatly reduce virus fusion, but only in the absence of a prebinding step; they had no effect if this step was included. Although membrane merging was greatly reduced following cell neuraminidase pretreatment, virus-cell association at low pH was not disturbed proportionately. This probably reflects unspecific virus-cell binding under these conditions, probably of inactivated or aggregated virus particles, which does not translate into membrane merging. This seems to suggest both that target membrane sialic acid can protect the virus from losing its activity before triggering membrane merging, and that the importance of this interaction is not merely to ensure virus-target proximity. With liposomes, we found that both types of ganglioside supported efficient fusion, with GD1a promoting a slightly faster initial rate. However, in this case, virus-target proximity closely mirrored fusion activity, thus pointing to differential specificity between targets routinely used to assay influenza virus fusion activity.     

Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion.

To investigate the function of the envelope glycoproteins gp50 and gII of pseudorabies virus in the entry of the virus into cells, we used linker insertion mutagenesis to construct mutant viruses that are unable to express these proteins. In contrast to gD mutants of herpes simplex virus, gp50 mutants, isolated from complementing cells, were able to form plaques on noncomplementing cells. However, progeny virus released from these cells was noninfectious, although the virus was able to adsorb to cells. Thus, the virus requires gp50 to penetrate cells but does not require it in order to spread by cell fusion. This finding indicates that fusion of the virus envelope with the cell membrane is not identical to fusion of the cell membranes of infected and uninfected cells. In contrast to the gp50 mutants, the gII mutant was unable to produce plaques on noncomplementing cells. Examination by electron microscopy of cells infected by the gII mutant revealed that enveloped virus particles accumulated between the inner and outer nuclear membranes. Few noninfectious virus particles were released from the cell, and infected cells did not fuse with uninfected cells. These observations indicate that gII is involved in several membrane fusion events, such as (i) fusion of the viral envelope with the cell membrane during penetration, (ii) fusion of enveloped virus particles with the outer nuclear membrane during the release of nucleocapsids into the cytoplasm, and (iii) fusion of the cell membranes of infected and uninfected cells.     

The C- and the N-terminal regions of glycoprotein 41 ectodomain fuse membranes enriched and not enriched with cholesterol, respectively

To infect target cells, HIV-1 employs a virally encoded transmembrane protein (gp41) to fuse its viral envelope with the target cell plasma membrane. We describe the gp41 ectodomain as comprised of N- and C-terminal subdomains, each containing a heptad repeat as well as a fusogenic region, whose organization is mirrored by the intervening loop region. Recent evidence indicates that the gp41 directed fusion reaction proceeds to initial pore formation prior to gp41 folding into its low energy hairpin conformation. This implies that exposed regions of the gp41 ectodomain are responsible for the bulk of the fusion work, probably through direct protein-membrane interactions. Prevalent fusion models contend that the gp41 ectodomain initially interacts with the target cell surface through its highly hydrophobic N terminus, which is believed to insert into the target membrane, thereby linking the virus to the target cell. This arrangement allows the N-terminal subdomain to interact with the target cell surface, whereas the C-terminal subdomain remains proximal to the virion, allowing interaction with the viral envelope. The composition of the viral envelope and the target cell surface differ due to the virus budding from raft microdomains. We show here that constructs corresponding to the C-terminal subdomain specifically destabilize ordered and cholesterol rich membranes (33 molar %), whereas the N-terminal subdomain is more effective in fusing both unordered cholesterol-free membranes and those containing lower amounts of cholesterol (10 molar %). Moreover we show that, in the context of the C-terminal subdomain, the heptad repeat contributes helical structure, which may describe the enhanced inhibitory effect of the C-terminal subdomain relative to the C-terminal heptad repeat (C34) alone. Our results are discussed in light of recent findings that showcase the role of exposed gp41 regions in effecting membrane fusion.     

Receptor-induced thiolate couples Env activation to retrovirus fusion and infection

According to current models of retrovirus infection, receptor binding to the surface subunit (SU) of the envelope glycoprotein (Env) triggers a conformational change in the transmembrane subunit (TM) that mediates virus fusion to cell membranes. To understand how this occurs, we investigated the role of the receptor Tva in avian leukosis virus-A (ALV-A) infection. We find that Tva binding induced the formation of a reactive thiolate on Cys38 (Cys38-S- in SU. Both chemical and genetic inactivation of Cys38-S- completely abrogated ALV fusion and infection. Remarkably, Cys38-S- does not mediate isomerization of the SU-TM disulfide bond and is not required for Tva-induced activation of TM, including pre-hairpin association with membranes and low pH assembly of helical bundles. These findings indicate that, contrary to current models, receptor activation of TM is not sufficient for ALV fusion and infection and that formation of a reactive thiolate is an additional receptor-dependent step.     

Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: stability and pH effects

Ebola virus (EboV) belongs to the Filoviridae family of viruses that causes severe and fatal hemhorragic fever. Infection by EboV involves fusion between the virus and host cell membranes mediated by the envelope glycoprotein GP2 of the virus. Similar to the envelope glycoproteins of other viruses, the central feature of the GP2 ectodomain postfusion structure is a six-helix bundle formed by the protein's N- and C-heptad repeat regions (NHR and CHR, respectively). Folding of this six-helix bundle provides the energetic driving force for membrane fusion; in other viruses, designed agents that disrupt formation of the six-helix bundle act as potent fusion inhibitors. To interrogate determinants of EboV GP2-mediated membrane fusion, we designed model proteins that consist of the NHR and CHR segments linked by short protein linkers. Circular dichroism and gel filtration studies indicate that these proteins adopt stable α-helical folds consistent with design. Thermal denaturation indicated that the GP2 six-helix bundle is highly stable at pH 5.3 (melting temperature, T(m) , of 86.8 ± 2.0°C and van't Hoff enthalpy, ΔH(vH) , of -28.2 ± 1.0 kcal/mol) and comparable in stability to other viral membrane fusion six-helix bundles. We found that the stability of our designed α-helical bundle proteins was dependent on buffering conditions with increasing stability at lower pH. Small pH differences (5.3-6.1) had dramatic effects (ΔT(m) = 37°C) suggesting a mechanism for conformational control that is dependent on environmental pH. These results suggest a role for low pH in stabilizing six-helix bundle formation during the process of GP2-mediated viral membrane fusion.     

Fusion promotion by a paramyxovirus hemagglutinin-neuraminidase protein: pH modulation of receptor avidity of binding sites I and II

Paramyxoviruses, including the childhood respiratory pathogen human parainfluenza virus type 3 (HPIV3), possess an envelope protein hemagglutinin-neuraminidase (HN) that has receptor-cleaving (neuraminidase), as well as receptor-binding, activity. HN is a type II transmembrane glycoprotein, present on the surface of the virus as a tetramer composed of two dimers. HN is also essential for activating the fusion protein (F) to mediate merger of the viral envelope with the host cell membrane. This initial step of viral entry occurs at the host cell surface at neutral pH. The HN molecule carries out these three different critical activities at specific points in the process of viral entry, and understanding the regulation of these activities is key for the design of strategies that block infection. One bifunctional site (site I) on the HN of HPIV3 possesses both receptor binding and neuraminidase activities, and we recently obtained experimental evidence for a second receptor binding site (site II) on HPIV3 HN. Mutation of HN at specific residues at this site, which is next to the HN dimer interface, confers enhanced fusion properties, without affecting neuraminidase activity or receptor binding at neutral pH. We now demonstrate that mutations at this site II, as well as at site I, confer pH dependence on HN's receptor avidity. These mutations permit pH to modulate the binding and fusion processes of the virus, potentially providing regulation at specific stages of the viral life cycle.     

A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion

The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.     

Functional Analysis of the Autographa californica Multiple Nucleopolyhedrovirus GP64 Terminal Fusion Loops and Interactions with Membranes

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) glycoprotein GP64 is the major envelope protein of the budded virus (BV). GP64 is a class III fusion protein that mediates BV attachment to the cell surface and low-pH-triggered membrane fusion between the BV envelope and the endosome membrane during entry. Class III fusion proteins contain terminal looped structures that are believed to interact with membranes. To examine the functions of 3 loops found at the apex of the GP64 postfusion structure, we generated 2-alanine substitutions that scanned the two so-called fusion loops (loop 1 and loop 2) plus an adjacent loop structure (loop 3) that is closely attached to loop 2 and is also found at the apex of the GP64 postfusion structure. We identified essential residues from Y75 to T86 (loop 1) and N149 to H156 (loop 2) that are required for fusion activity, but no essential residues in loop 3. Further analysis revealed that critical fusion loop residues fall within two groups that are associated with either membrane merger (hemifusion) or fusion pore expansion. We next examined the interactions of soluble GP64 proteins and BV with membranes composed of various phospholipids. BV interacted directly with small unilamellar vesicles (SUVs) comprised of phospholipids phosphatidylcholine and phosphatidic acid (PC/PA) or phosphatidylcholine and phosphatidylserine (PC/PS) under neutral and acidic pH. We also examined the interactions of soluble GP64 constructs containing substitutions of the most hydrophobic residues within each of the two fusion loops. We found that a 2-residue substitution in either single loop (loop 1 [positions 81 and 82] or loop 2 [positions 153 and 154]) was not sufficient to substantially reduce the GP64-liposome interaction, but the same substitutions in both fusion loops severely reduced the GP64-liposome association at neutral pH. These results suggest that critical hydrophobic residues in both fusion loops may be involved in the interaction of GP64 with host cellular membranes and direct GP64-membrane interactions may represent a receptor-binding step prior to a low-pH-triggered conformational change.     

Inhibition of murine leukemia virus envelope protein (env) processing by intracellular expression of the env N-terminal heptad repeat region

A conserved structural motif in the envelope proteins of several viruses consists of an N-terminal, alpha-helical, trimerization domain and a C-terminal region that refolds during fusion to bind the N-helix trimer. Interaction between the N and C regions is believed to pull viral and target membranes together in a crucial step during membrane fusion. For several viruses with type I fusion proteins, C regions pack as alpha-helices in the grooves between N-helix monomers, and exogenously added N- and C-region peptides block fusion by inhibiting the formation of the six-helix bundle. For other viruses, including influenza virus and murine leukemia virus (MLV), there is no evidence for comparably extended C-region alpha-helices, although a short, non-alpha-helical interaction structure has been reported for influenza virus. We tested candidate N-helix and C-region peptides from MLV for their ability to inhibit cell fusion but found no inhibitory activity. In contrast, intracellular expression of the MLV N-helix inhibited fusion by efficiently blocking proteolytic processing and intracellular transport of the envelope protein. The results highlight another mechanism by which the N-helix peptides can inhibit fusion.     

Low pH is required for avian sarcoma and leukosis virus Env-induced hemifusion and fusion pore formation but not for pore growth

Binding of avian sarcoma and leukosis virus (ASLV) to its cognate receptor on the cell surface causes conformational changes in its envelope protein (Env). It is currently debated whether low pH is required for ASLV infection. To elucidate the role of low pH, we studied the association between ASLV subgroup B (ASLV-B) and liposomes and fusion between effector cells expressing Env from ASLV-A and ASLV-B and target cells expressing cognate receptors. Neither EnvA nor EnvB promoted cell-cell fusion at neutral pH, but lowering the pH resulted in quick and extensive fusion. As expected for a low-pH-triggered reaction, fusion was a steep function of pH. Steps that required low pH were identified. Binding a soluble form of the receptor caused ASLV-B to hydrophobically associate with liposome membranes at neutral pH, indicating that low pH is not required for insertion of Env's fusion peptides into membranes. But both cell-cell hemifusion and fusion pore formation were pH dependent. It is proposed that fusion peptide insertion stabilizes the conformation of ASLV Env into a form that can be acted upon by low pH. At this point, but not before, low pH can induce fusion and is in fact required for fusion to occur. However, low pH is no longer necessary after formation of the initial fusion pore: pore enlargement does not require low pH.