Cholesterol is required in the exit pathway of Semliki Forest virus

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.     

Differential cholesterol binding by class II fusion proteins determines membrane fusion properties

The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.     

Role of spike protein conformational changes in fusion of Semliki Forest virus.

The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.     

The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of approximately 100-fold in infection and approximately 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.     

Biochemical consequences of a mutation that controls the cholesterol dependence of Semliki Forest virus fusion

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion of srf-3 and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast, srf-3 fusion with sterol-free liposomes was significantly more efficient than that of wt virus. Thus, the srf-3 mutation does not affect its general fusion properties with purified lipid bilayers but causes a marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol dependent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.     

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.     

Membrane fusion mutants of Semliki Forest virus

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild- type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.     

Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids

Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent on the presence of cholesterol in the target membrane. In this paper, we show that fusion of SFV with cholesterol-containing liposomes depends on sphingomyelin (SM) or other sphingolipids in the target membrane, ceramide representing the sphingolipid minimally required for mediating the process. The action of the sphingolipid is confined to the actual fusion event, cholesterol being necessary and sufficient tor low-pH-dependent binding of the virus to target membranes. The 3-hydroxyl group on the sphingosine backbone plays a key role in the SFV fusion reaction, since 3-deoxy-sphingomyelin does not support the process. This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1–2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a co-factor, possibly through activation of the viral fusion protein. Domain formation between cholesterol and sphingolipid, although it may facilitate SFV fusion, is unlikely to play a crucial role in the process.     

Penetration of Semliki Forest virus from acidic prelysosomal vacuoles

To identify and characterize the intracellular site from which the penetration of Semliki Forest virus (SFV) to the cytosolic compartment of the host cell occurs, we determined the time course and temperature dependence of nucleocapsid uncoating and infection in BHK-21 cells. At 37 degrees C the genome release to the cytosol was detected within 5-7 min after virus endocytosis, whereas delivery of the virus particles to secondary lysosomes occurred within 15-20 min. At temperatures of 15 degrees -20 degrees C virus particles were internalized by endocytosis, but they were not delivered to the secondary lysosomes. Nevertheless, at 20 degrees C nucleocapsid uncoating and infection occurred, indicating that secondary lysosomes are not required for SFV penetration. We conclude that the penetration reaction occurs in prelysosomal endocytic vacuoles (endosomes). As SFV penetration by membrane fusion requires a pH less than 6 and the presence of cholesterol in the target membrane, the data indicate that endosomes are acidic and contain cholesterol.     

On the entry of semliki forest virus into BHK-21 cells

The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.     

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.     

The fusion peptide of Semliki Forest virus associates with sterol-rich membrane domains

Semliki Forest virus (SFV) is an enveloped alphavirus whose membrane fusion is triggered by low pH and promoted by cholesterol and sphingolipid in the target membrane. Fusion is mediated by E1, a viral membrane protein containing the putative fusion peptide. Virus mutant studies indicate that SFV's cholesterol dependence is controlled by regions of E1 outside of the fusion peptide. Both E1 and E1*, a soluble ectodomain form of E1, interact with membranes in a reaction dependent on low pH, cholesterol, and sphingolipid and form highly stable homotrimers. Here we have used detergent extraction and gradient floatation experiments to demonstrate that E1* associated selectively with detergent-resistant membrane domains (DRMs or rafts). In contrast, reconstituted full-length E1 protein or influenza virus fusion peptide was not associated with DRMs. Methyl beta-cyclodextrin quantitatively extracted both cholesterol and E1* from membranes in the absence of detergent, suggesting a strong association of E1* with sterol. Monoclonal antibody studies demonstrated that raft association was mediated by the proposed E1 fusion peptide. Thus, although other regions of E1 are implicated in the control of virus cholesterol dependence, once the SFV fusion peptide inserts in the target membrane it has a high affinity for membrane domains enriched in cholesterol and sphingolipid.     

A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.     

Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell- cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.     

In vivo generation and characterization of a soluble form of the Semliki forest virus fusion protein

During infection of host cells, a number of enveloped animal viruses are known to produce soluble forms of viral membrane glycoproteins lacking the transmembrane domain. The roles of such soluble glycoproteins in viral life cycles are incompletely understood, but in several cases they are believed to modulate host immune response and viral pathogenesis. Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells through low-pH-dependent fusion and buds from the plasma membrane. Fusion is mediated by the E1 subunit of the SFV spike protein. Previous studies described the in vivo generation of E1s, a truncated soluble form of E1, under conditions in which budding is inhibited in mammalian host cells. We have here examined the properties of E1s generation and the biological activity of E1s. E1s cleavage required spike protein transport out of the endoplasmic reticulum and was independent of virus infection. Cell surface E1 efficiently acted as a precursor for E1s. E1s generation was strongly pH dependent in BHK cells, with optimal cleavage at a pH of < or =7.0, conditions that inhibited the budding of SFV but not the budding of the rhabdovirus vesicular stomatitis virus. The pH dependence of E1s production and SFV budding was unaffected by the stability of the spike protein dimer but was a function of the host cell. Similar to the intact virus and in vitro-generated E1 ectodomain, treatment of E1s at low pH in the presence of target membranes triggered specific acid-dependent conformational changes. Thus, under a variety of conditions, SFV-infected cells can produce a soluble form of E1 that is biologically active.     

Alphavirus Restriction by IFITM Proteins

Interferon inducible transmembrane proteins (IFITMs) are broad‐spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo‐, flavi‐, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid‐induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH‐induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV.      

Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein.

Semliki Forest virus (SFV), an alphavirus, infects cells via a low pH-triggered membrane fusion reaction that takes place within the cellular endocytic pathway. Fusion is mediated by the heterotrimeric virus spike protein, which undergoes conformational changes upon exposure to low pH. The SFV E1 spike subunit contains a hydrophobic domain of 23 amino acids that is highly conserved among alphaviruses. This region is also homologous to a domain of the rotavirus outer capsid protein VP4. Mutagenesis of an SFV spike protein cDNA was used to evaluate the role of the E1 domain in membrane fusion. Mutant spike proteins were expressed in COS cells and assayed for cell-cell fusion activity. Four mutant phenotypes were identified: (i) substitution of Gln for Lys-79 or Leu for Met-88 had no effect on spike protein fusion activity; (ii) substitution of Ala for Asp-75, Ala for Gly-83, or Ala for Gly-91 shifted the pH threshold of fusion to a more acidic range; (iii) mutation of Pro-86 to Asp, Gly-91 to Pro, or deletion of amino acids 83 to 92 resulted in retention of the E1 subunit within the endoplasmic reticulum; and (iv) substitution of Asp for Gly-91 completely blocked cell-cell fusion activity without affecting spike protein assembly or transport. These results argue that the conserved hydrophobic domain of SFV E1 is closely involved in membrane fusion and suggest that the homologous region in rotavirus VP4 may be involved in the entry pathway of this nonenveloped virus.     

Fusion of Semliki forest virus with the plasma membrane can be induced by low pH

When BHK-21 cells with Semliki Forest virus (SFV) bound at the plasma membrane are briefly treated with low pH medium (pH 5-6), fusion between the viral membrane and the plasma membrane occurs, releasing the viral nucleocapsid into the cytoplasm. The fusion reaction resembles that described previously for Sendai virus but with one fundamental difference; it is strictly dependent on low pH. The fusion reaction is highly efficient. Up to 86% of bound viruses fuse, and 6 X 10(6) virus spike proteins can be inserted into the plasma membrane of each cell. The process is very rapid (full activity is observed after 5 s) and it occurs over a wide temperature range and equally well with all five cell lines tested (BHK-21, HeLa B, HeLa suspension, Raji, and 3T3). Low pH-induced fusion of the virus at the plasma membrane can lead to infection of susceptible cells. The artificial nature of this infection pathway is, however, demonstrated by the facts that infection through the plasma membrane occurs only at subphysiological pH and that it is insensitive to inhibitors of the normal entry route. Nevertheless, these results indicate that low pH membrane fusion introduces the viral genome into the cytoplasm in a form suitable for replication.     

Sphingolipid and cholesterol dependence of alphavirus membrane fusion. Lack of correlation with lipid raft formation in target liposomes

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes. Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane. This is suggestive of a possible involvement of sphingolipid-cholesterol microdomains, or "lipid rafts," in the membrane fusion and cell entry process of the virus. In this study, large unilamellar vesicles (LUVs) were prepared from synthetic sphingolipids and sterols that vary with respect to their capacity to promote microdomain formation, as assessed by gradient flotation analysis in the presence of Triton X-100. SFV and SIN fused with LUVs irrespective of the presence or absence of Triton X-100-insoluble microdomains. These results suggest that SFV and SIN do not require the presence of lipid rafts for fusion with target membranes. Furthermore, it is not necessary for sphingolipids to reside in a detergent-insoluble complex with cholesterol to promote SFV or SIN fusion.     

Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell

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.