Role of the Cytoplasmic Tail of Ecotropic Moloney ... - Journal of Virology

JOURNAL OF VIROLOGY, Jan. 2000, p. 447–455 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 1

Role of the Cytoplasmic Tail of Ecotropic Moloney Murine Leukemia Virus Env Protein in Fusion Pore Formation GRIGORY B. MELIKYAN,1 RUBEN M. MARKOSYAN,1 SOFYA A. BRENER,1 YANINA ROZENBERG,2 AND FREDRIC S. COHEN1* Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612,1 and Gene Therapy Laboratories, Norris Cancer Center, University of Southern California School of Medicine, Los Angeles, California 900332 Received 14 June 1999/Accepted 20 September 1999

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*—a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding—resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4°C and the time until fusion measured after the temperature was raised to 37°C or whether cells were first brought into contact at 37°C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion. receptor, which occurs at neutral pH, triggers the conformational changes in the Env protein, which allows fusion to proceed. Mo-MuLV is different than most other enveloped viruses in that the fusogenic activity of its Env protein is controlled by the trimming of the protein’s cytoplasmic tail (CT). When synthesized, the CT of the p15E subunit is 32 amino acid residues long (residues 601 to 632). At the time Mo-MuLV buds from the cell, the 16 C-terminal residues (referred to as the R peptide) of the CT are removed by a viral protease (10, 16, 36), greatly increasing the fusogenic ability of Env (18, 33, 35). The role of the length of the CT in Mo-MuLV Env-induced fusion has been established by expressing the fusion protein with full-length and truncated CTs in cells and testing their ability to form heterokaryons (i.e., syncytia) with target cells containing ecotropic virus receptors. Cells expressing Env with a CT truncated by 16 amino acid residues (“R-less” or Env 616*) have the highest syncytial potency (18, 33, 35). Deleting the CT altogether to residue 601 (Env 601*) leads to a somewhat reduced extent of syncytia formation, while further truncation that removes residues from the C-terminal portion of the membrane-spanning (MS) domain (e.g., Env 595*) completely abolishes polykaryon formation (Y. Rozenberg et al., submitted for publication). For syncytia to form, not only must fusion occur, but other events, such as major pore growth, cytoskeleton rearrangements, and movement of nuclei, must proceed as well. Therefore, additional experimental approaches are required to delineate the process of fusion pore formation and its enlargement. We have used fluorescence microscopy to monitor lipid continuity and electrophysiological measurements to record fusion pores in order to assess the effect of the CT of Env on membrane fusion and on the early stages of pore growth. We show

The Env protein of ecotropic Moloney murine leukemia virus (Mo-MuLV) is a homotrimeric bifunctional protein responsible for binding to host receptor and fusion of the envelope with the cell plasma membrane (15, 28, 41, 44). Each monomer of the Env protein is synthesized as a gp85 precursor, which is posttranslationally cleaved by cellular proteases into gp70 (surface; SU) and p15E (transmembrane; TM) subunits (29) which are responsible for binding and fusion respectively. The core structure of Mo-MuLV Env is strikingly similar to those of other viral fusion proteins. Fusion proteins for Mo-MuLV (15), human immunodeficiency virus (5, 43), influenza virus (4), Ebola virus (42), and SV5 (a paramyxovirus) (2) contain a triple coiled-coil core surrounded by three C-terminal ␣-helices running antiparallel to the central stem. The presence of common structural features suggests that different viral fusion proteins induce membrane merger by similar mechanisms. The mechanism of fusion has been most extensively delineated for the hemagglutinin (HA) of influenza virus, which thus serves as a prototypic fusion protein (17). Based on the similarity of the crystallographic structures of their TM subunits, it is expected that, analogous with HA, conformational changes in the TM subunits of Mo-MuLV Env lead to an extended coiled-coil stem region and insertion of the subunits’ fusion peptides into the target membrane. In a manner not fully understood, this causes fusion between the MoMuLV envelope and the plasma membrane to which it is bound. Fusion of Mo-MuLV proceeds at neutral pH (32, 35). It is believed that the binding of the SU subunit to its specific * Corresponding author. Mailing address: Department of Molecular Biophysics and Physiology, Rush Medical College, 1653 W. Congress Pkwy., Chicago, IL 60612. Phone: (312) 942-6753. Fax: (312) 942-8711. E-mail: [email protected]. 447

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FIG. 1. Mo-MuLV Env constructs. The asterisk indicates a stop codon terminating the CT sequence after the indicated residue number. MelR⫺ is a chimeric construct wherein the CT was derived from melittin, a membrane-active amphipathic peptide. The vertical dotted line shows the division of the MS domain and beginning of the CT. The precise location where the MS domain ends and the CT begins is not known. By standard considerations of hydrophobicity, the CT would begin at Arg601. However, the sequence Gly595-Pro596 might introduce a turn and initiate an amphiphilic ␣-helix that runs from residues 598 to 616 and that functions as a unit (Rozenberg et al., submitted; see also below). In our descriptions, we retain the conventional division between the MS domain and the CT based on hydrophobicity but remain cognizant of a possible unity of function of the residue 595 to 616 region. Whereas residues 595 to 600 are part of the MS domain, based on hydrophobicity, it has been argued that this region is in fact part of the CT and is therefore also referred to as the “membrane-proximal region” (Rozenberg et al., submitted).

that deleting the C-terminal R peptide from the full-length CT (yielding Env 616*) strongly promoted a very early step required for syncytia: formation of the fusion pore itself. Deletion of almost the entire CT (Env 601*) had little further effect on fusion pore formation or on the initial size or growth of the pore. Additional truncation of six C-terminal residues (up to G595) from the predicted MS domain completely abolished fusion. A chimera consisting of residues 1 to 599 of the Env of Mo-MuLV followed by a CT, consisting of the amphiphilic portion of melittin, induced fusion pores similar to those caused by Env 601* and Env 616*. We conclude that the CT itself is not needed for Env-induced fusion but that residues 596 to 601 are required (either directly or indirectly through affecting other portions of Env) for fusion. MATERIALS AND METHODS Envelope constructs, cell culture, and Env expression. The envelope protein constructs were produced as described elsewhere (Rozenberg et al., submitted). An asterisk denotes that a stop codon follows the indicated residue number (Fig. 1). The expressed Env protein is denoted by its C-terminal residue number (e.g., 616* denotes the Env protein consisting of residues 1 to 616). MelR⫺ denotes a chimera between residues 1 and 599 of Mo-MuLV Env and a fragment of melittin (serving as a CT; Fig. 1). The “M” stands for methionine, a mutation inadvertently introduced during PCR mutagenesis. This position is normally occupied by isoleucine. The XC6 cell line (a highly fusion permissive subclone of XC rat sarcoma cells [Y. Rozenberg, unpublished observations]) and NIH 3T3 fibroblasts were grown within a humidified 5% CO2 incubator in basal minimal essential medium or Dulbecco modified Eagle medium (DMEM), respectively, supplemented with 10% Cosmic Calf Serum (HyClone Laboratories, Logan, Utah), L-glutamine, and penicillin-streptomycin (GIBCO BRL, Gaithersburg, Md.). HEK 293T (referred to as 293T) cells were maintained in DMEM-Cosmic Serum supplemented with 0.5 mg of geneticin per ml. This cell line has excellent transfection efficiency and lacks ecotropic Env receptors, which precludes cellcell fusion among themselves even though the Env protein is expressed on their surfaces. Different constructs of the Env protein of Mo-MuLV were transiently expressed in 293T cells by transfecting with the plasmid pHIT123 (37) by using calcium phosphate. A total of 15 ␮g of Env 632*, 601*, 595*, and MelR⫺ plasmids and 12 ␮g of Env 616* were used per each 6-cm culture dish. Cells were incubated with a DNA precipitate for 4 h at 37°C in the presence of 25 ␮g of chloroquine per ml. Relative levels of surface expression of Env constructs were assessed by flow cytometry 48 h after transfection by using an anti-gp70 rat monoclonal antibody, 83A25, against Env protein as described previously (18). Labeling the cells with fluorescent dyes. The rationales for procedures used to fluorescently label cells have been described (9). In the current series of experiments, 293T cells expressing Env protein (defined as the “effector cells”) were

J. VIROL. labeled with the cytoplasmic marker CalceinAM (CaAM; Molecular Probes, Eugene, Oreg.). Cells were lifted from a 6-cm culture dish with phosphatebuffered saline (PBS) supplemented with 0.5 mM EDTA, 0.5 mM EGTA, and 3 mg of glucose per ml and labeled with 2 to 4 ␮M CaAM according to the manufacturer’s instructions. XC6 and NIH 3T3 (referred to as 3T3) fibroblasts (the “target cells”) were colabeled with the cytoplasmic marker 7-amino-4chloromethylcoumarin (CMAC; Molecular Probes, Inc.) and a lipophilic probe, either PKH-26 (Sigma Chemical Co., St. Louis, Mo.) or DiI (Molecular Probes). A confluent monolayer of target cells in a 10-cm culture dish (ca. 107 cells) was washed twice with PBS, and incubated in OptiMEM (GIBCO BRL) containing 40 ␮M CMAC for 30 min at 37°C. Cells were then incubated with dye-free OptiMEM for 15 min, lifted by a brief exposure to a trypsin-EDTA solution, resuspended in DMEM containing 10% bovine serum, and washed twice with PBS. CMAC-loaded cells were subsequently labeled with 1 to 2 ␮l of a 1 mM stock solution of PKH26 in ethanol or with 1 to 2 ␮g of DiI. Membrane dyes were injected into PBS, vortexed, briefly sonicated, and immediately mixed with an equal volume of cell suspension. Double-dye-labeled cells were washed once in DMEM supplemented with 10% bovine serum and then twice in PBS. Fluorescence video microscopy. The fluorescence of CaAM-labeled 293T cells was monitored by using a standard fluorescein filter set for an Axiovert 100A microscope (Carl Zeiss, Inc., Thornwood, N.Y.). The fluorescence of CMACPKH26-colabeled target cells was monitored by standard DAPI (4⬘,6⬘-diamidino2-phenylindole) and rhodamine filter sets, respectively. The amount of fluorescent dyes used to label the cells and the spectral characteristics of filter cubes was selected to minimize a bleed-through of fluorescence from one dye when using a filter set of another. Fluorescence images were monitored with an intensified (KS1380; Video Scope, Washington, D.C.) charge-coupled device video camera (Dage 72, Michigan City, Ind.) and recorded onto S-VHS videotape. Images were digitized by a video frame-grabber (Meteor; Matrox Electronic Systems, Dorval, Quebec, Canada) and a PC-based computer. Images acquired for each fluorescent dye were pseudocolored according to their emission wavelength (red, green, or blue) and superimposed on each other by using locally written imaging software. Cell-cell binding and fusion. Plastic culture dishes (35 mm) were precoated with poly-L-lysine (Mr, 70,000 to 150,000; Sigma) according to manufacturer’s instructions to ensure that the cells attached to the dishes. An approximately equal number of effector and target cells were mixed, transferred into the culture dishes, and allowed to bind to each other for 1 h at 4°C in PBS. When fusion was quantified, the density of effector and target cells was made sufficiently low so that cell pairs still formed but larger aggregates did not. After coincubation at 4°C, cells were washed once to remove unattached cells, and fusion was induced by raising the temperature to 37°C for controlled times. A three-dye assay that scores cell-cell fusion by monitoring the spread of membrane and aqueous dye between effector and target cells was used (26). Several randomly selected fields were analyzed for each culture dish (more than 100 cell pairs were screened per dish). Bright-field images combined with fluorescence images were used to determine the total number of effector-target cell pairs. Cell pairs stained with all three fluorescent markers were scored as fused and normalized by the total number of pairs in the field. To quickly step from a nonpermissive-fusion temperature to 37°C, cells were brought under an infrared laser diode (Model A001-FC/100; Opto Power Corp., Tuscon, Ariz.). The laser diode output was set so that it melted eicosane (Sigma; melting point, 36 to 38°C) but not henecosane (melting point, 40 to 42°C) spread as a thin film over a glass coverslip placed in the aqueous buffer. The region of melted eicosane was about 300 ␮m in diameter, providing an estimate of the effective diameter of the infrared beam. The steady-state temperature was established within 2 to 4 s depending on the initial temperature of the bathing solution. Electrophysiological measurement of fusion pore formation. Fusion of an Env-expressing 293T cell to a target XC6 cell was monitored electrically in the whole-cell patch clamp configuration as an increase in electrical capacitance of the patched cell membrane due to the addition of the fused cell membrane (30, 31, 39). The full capacitance (i.e., area) of the target cell membrane is revealed only when the fusion pore connecting the effector and target cells is large. Conductances of small and intermediate fusion pores were calculated from the increment in whole-cell admittance as GP ⫽ (Y02 ⫹ Y902)/Y0, where Y0 and Y90 are the increases in the in-phase and 90° out-of-phase components of electrical admittance, respectively (34). For electrophysiological experiments, target and effector cells were mixed, adhered to a poly-L-lysine-coated coverglass in the cold, and stored on ice prior to the experiment. Patch clamp experiments were conducted at 37°C. A 293T cell (labeled with CaAM to allow easy identification) was patched and lifted from the coverglass after a high-resistance seal formed between the patch pipette and the cell. The 293T cell was then brought into contact with a solitary XC6 cell (either unlabeled or labeled with PKH26). At the moment of physical contact between the two cells, the time was set equal to zero.

RESULTS An assay for cell-cell fusion: tracking the movement of three dyes. Fusion was monitored between cells expressing Env proteins (defined as effector) and cells containing receptors for ecotropic Mo-MuLV Env (defined as target). Human 293T

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Mo-MuLV Env FUSION PORES

TABLE 1. Surface expression of Env proteinsa Env constructs

% Env-expressing cells (n)

Normalized mean fluorescence

632* (wild type) 616* 601* 595* MelR⫺

21 ⫾ 3 (11) 37 ⫾ 6 (11) 37 ⫾ 3 (12) 60 ⫾ 3 (7) 26 ⫾ 8 (5)

100 ⫾ 10 160 ⫾ 19 70 ⫾ 6 143 ⫾ 13 100 ⫾ 11

a The numbers in the second and third columns are means ⫾ standard errors. The numbers of experiments conducted for each construct are shown in parentheses.

cells, transiently transfected with a plasmid containing the desired Mo-MuLV Env construct, do not contain receptors recognized by Mo-MuLV Env and served as the effector cells. XC6 cells and 3T3 fibroblasts were used as target cells. All the Env constructs were expressed efficiently on surfaces of 293T cells as judged by the presence of Env epitopes detected by flow cytometry (Table 1). The expression of Env 601* was consistently lower but still within about a factor of 2 of the other constructs. Attempts to increase the expression level of Env 601* by using a greater amount of plasmid DNA for transfection were unsuccessful (not shown). Env 595* and Env 616* were expressed at higher densities than the full-length Env 632*. The fusion activities of the Env constructs were first assessed by fluorescence microscopy at 48 h posttransfection. The effector 293T cells were loaded with the cytoplasmic marker CaAM (green fluorescence). The target cells were loaded with the cytoplasmic marker CMAC (blue fluorescence) and the membrane label PKH26 (red fluorescence). Labeled cells were removed from their culture dishes, and then effector and target cells were brought into contact by coincubating them in polylysine-coated culture dishes at 4°C for ca. 1 h. By this time, most cells were firmly adhered to the dishes, and some cells formed contacts with each other. The density of plated cells on the culture dish influenced whether cell pairs or larger aggregates between effector and target formed. To qualitatively determine the fusion potency of an Env construct, target and effector cells were densely seeded on culture dishes. Fusion was triggered by raising the temperature to 37°C for controlled times (usually between 10 and 60 min). Upon cell-cell fusion, both aqueous dyes (CMAC and CaAM) and the membrane probe redistributed. Consequently, the effector and target cells became stained by all three fluorescent dyes (Fig. 2). Fusion only occurred between effector and target cells since the effector 293T cells lack the receptors for the murine ecotropic viral Env. Fluorescent images of the three probes were acquired sequentially and superimposed on each other, so that the final color of the fused cells was the sum of the red, blue, and green colors scaled by their relative intensities. The additive mixing of these colors resulted in the fused cells appearing to be spectrally white, as displayed by computer (Fig. 2, arrows). (There were also occasional white spots due to effector and target cells that had not fused but instead rested on top of each other.) Cells expressing Env 632* (unclipped, full-length CT) did not fuse to 3T3 fibroblasts (Fig. 2, left panels) and fused to only a few XC6 cells after a 10-min incubation at 37°C (Fig. 2, right panels). In contrast, fusion was so extensive with Env 616* (arrowheads), for both 3T3 and XC6 as target cells, that irregularly shaped small syncytia formed. The Env 601* construct also induced fusion (arrows), but fusion did not usually extend beyond two cells: the whitish areas were noticeably smaller than those formed by Env 616*-expressing cells. Env 595* was

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completely inactive for both target cells. Env 632* and Env 595* were unable to induce even hemifusion to 3T3 cells, as indicated by a lack of PKH26 redistribution. Chlorpromazine (CPZ) is a membrane-permeable cationic drug that promotes full fusion between hemifused cell-erythrocyte pairs (22). Applying 0.4 mM CPZ after incubation of Env-expressing and target cells at 37°C did not improve fusion efficiency for either the 632* or 595* constructs (not shown). This finding further supports the conclusion that these constructs do not induce hemifusion. Env-mediated fusion is more efficacious with XC6 cells than with NIH 3T3 cells as the target. We compared the ability of two cell lines, NIH 3T3 and XC6 cells, to fuse to human 293T expressing Env constructs. 293T cells expressing an Env (that supported fusion) had to be incubated with 3T3 cells at 37°C for at least 25 min for the fluorescent dye to redistribute; with a 60-min incubation, the dye redistribution was complete. In contrast, with XC6 cells as targets, fusion occurred as early as 2 to 3 min after an increase of the temperature to 37°C. (These observations dictated the choice of the time points in Fig. 2.) These results are consistent with the previously observed higher Env-induced syncytium-forming activity for XC cells than for 3T3 fibroblasts as targets (18, 21). Fusion was so efficient that even when temperatures were kept relatively low, between 16 and 23°C, some Env 616*-expressing 293T cells fused to XC6 cells within ca. 1 h. Thus, the fusion step alone could account for the higher level of observed syncytium formation with XC cells; there need not be any differences in the further steps required for syncytia to form, such as extensive pore enlargement. Env 616* and Env 601*, but not Env 595* and Env 632*, were capable of inducing substantial fusion. In order to quantify the extent of fusion for the Env proteins with CTs truncated to differing extents, cells were plated at lower densities so that cell pairs, rather than aggregates, were preferentially formed. The fraction of effector-target cell pairs that were stained with all three fluorescent dyes provided the extent of fusion. The quantitative level of fusion for the various forms of Env (Fig. 3) agreed with the representative images shown (Fig. 2). After a 1-h incubation of effector cells with 3T3 target cells at 37°C, the full-length CT (Env 632*) did not result in fusion (Fig. 3A). In contrast, virtually all cells expressing Env 616* fused to their bound target cells. Truncation of the CT to 601 (i.e., Env 601*) resulted in only a minor reduction of fusion activity, whereas a further truncation of six more amino acid residues (Env 595*) abolished fusion. The chimera consisting of residues 1 to 599 of Env, followed by the amphiphilic portion of the peptide melittin (Env MelR⫺), supported efficient fusion. With XC6 cells as target (Fig. 3B), the extent of fusion for each Env protein was similar to that observed with 3T3 cells (except for Env 632*), but for each construct (that supported fusion) fusion was quicker with XC6 cells. With only a 10-min incubation at 37°C (Fig. 3B, striped bars), almost all Env 616*expressing cells fused to their XC6 neighbors, whereas the 632* and 595* Env proteins were essentially inactive. The Env 601* protein induced fusion at levels comparable to those for MelR⫺. A longer (40-min) incubation at 37°C resulted in significant increases in the extent of fusion for the Env 601* and Env MelR⫺ constructs (Fig. 3B, open bars). At this time, Env 601* and Env MelR⫺ exhibited only slightly lower extents of fusion than did Env 616*. The order of extent of fusion between constructs was also reflected in the time it took for them to fuse to XC6 cells. Env 616* induced fusion in about 95% of the cell pairs within a few minutes at 37°C, while the maximal fusion induced by Env 601*

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took much longer (between 10 and 40 min). Env MelR⫺ exhibited the same extents of fusion as Env 601* at both 10 and 40 min. XC6 cells were such an effective target that Env 632* exhibited fusion at 40 min (Fig. 3B, open bar), with more than 20% of the bound cell pairs fused. This finding is consistent with the ability of Env 632* to promote syncytia with XC, but not 3T3, cells (Rozenberg et al., submitted). Notably, even a prolonged incubation of Env 595*-expressing cells with XC6 cells at 37°C for several hours did not result in measurable dye redistribution. Residues 596 to 601, missing in the Env 595* construct, are clearly important for fusion. Mo-MuLV Env-mediated fusion to XC6 cells is fast. In order to determine the fusion kinetics at early times after creating the conditions that permit fusion, Env-expressing 293T cells and XC6 cells were preincubated at 4°C for 1 to 2 h, and then the temperature was quickly stepped from 4 to 37°C. The lag times from raising the temperature to the onset of calcein redistribution from 293T to XC6 cells were monitored by video microscopy and plotted as cumulative distributions (Fig. 4A). These distributions provide the kinetics of fusion pores that have formed and grown large enough to allow the small molecule calcein (Mr, ⬃600) to pass through them. We used this temperature-raising method to continuously monitor the movement of dye spread between individual cell pairs for as long as 4 to 5 min. As cells that would have fused later were excluded, the distributions of waiting times to pore formation were truncated. For all three constructs tested, there was a delay before fusion: these distributions displayed an “S-shape” (rather than, for example, a hyperbolic rise without delay). The maximal rate of fusion induced by Env 616* was only about three times greater than that of Env 601* (Fig. 4). However, in addition, a smaller fraction of cells expressing Env 601* fused (27%) within 5 min than cells expressing Env 616* (72%). The slower rates and lesser extent of fusion over 5 min for Env 601* may simply reflect its lower level of expression (Table 1). The rapidity of fusion after raising the temperature to 37°C is underscored by noting that half of the Env 616*-expressing cells that did fuse over a 5-min period did so within 30 s (Fig. 4A). Neither Env 632* nor Env 595* formed fusion pores for periods as long as 10 min after the temperature was stepped to 37°C. Calcein redistributed faster than the other two fluorescent markers used to label the cells. The lag times for CMAC transfer (not shown) were 2 to 2.5 times greater than for calcein, suggesting that the membrane-impermeable products of CMAC (i.e., those reacted with glutathione and cytoplasmic proteins [Molecular Probes catalog]) are substantially larger than calcein. The membrane dyes, PKH26 and DiI, tended to segregate and gave an uneven pattern of fluorescence in target cells (Fig. 2). This may be the reason these dyes transferred more slowly than calcein, particularly if fusion pores quickly enlarged to allow unrestricted passage of the aqueous dye. The kinetics of formation of fusion pores (Fig. 4B) and the pattern of their growth was also determined electrophysiologically for the Env 616* and 601* constructs. For these experiments, effector cells were patch clamped, and the whole-cell configuration was established. These cells were then brought into contact with target cells at 37°C. As fusion was induced


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without the prebinding step at low temperature, these kinetics depend on (mathematically, a convolution of) the binding of Env to specific receptors and the fusion reaction itself. Despite the fact that these electrophysiological measures of fusion would be delayed by binding steps, the kinetics of Env 601* and Env 616* (Fig. 4B) were similar to those obtained by measuring transfer of aqueous dye between prebound cells (Fig. 4A). Initial conductance and enlargement of small fusion pore formed by Env. Fusion pore behavior was analyzed for Env 616*, 601*, and MelR⫺ constructs by means of capacitance measurements in the whole-cell patch clamp configuration. Small fusion pores are the earliest detectable events in fusion. They establish cytoplasmic continuity and membrane merger. As readily seen from the representative traces (Fig. 5), all three constructs generated similar pores at their early stage of growth, regardless of the length (Env 616* versus Env 601*) and the sequence (Env 616* versus Env MelR⫺) of the CT. A fusion pore is not a static structure; its conductance varies from moment to moment (Fig. 5). Because the precise variation in conductance is different for every experiment, we characterized the fusion pores formed by Env 616*, Env 601*, and the chimeric Env MelR⫺ by averaging, for each construct, the conductances over time from all experiments (Fig. 6). The initial conductances of pores were statistically the same for these constructs (Fig. 6A). The initial pore induced by any of the three Env proteins was relatively large: pore conductance was about 2 nS or approximately 7 nm in diameter. The pores also grew readily. An open pore formed by Mo-MuLV Env almost never closed (a process termed flickering). Although at early times (Fig. 6A) the pores formed by Env 601* tended to reach somewhat larger conductance levels than the pores formed by the other two constructs, the differences were not statistically significant (see legend to Fig. 6). The estimated pore diameter exceeded 17 nm within the first 3 s of formation (not shown). Pore growth was steady and continuous for all three Env constructs (Fig. 6B); Env 601* promoted somewhat slower growth. Env 632* and Env 595* did not form fusion pores for as long as 10 min after establishment of cell-cell contact, in agreement with the aqueous dye spread measurements. DISCUSSION Fusion pores formed by retroviral Env protein are similar to those created by other viral proteins. This is the first study that has electrically characterized fusion pores created by a retroviral fusion protein. The nascent fusion pores formed by Env protein of Mo-MuLV have an average diameter of about 7 nm (conductance, ca. 2 nS). These sizes are similar to those of pores formed by baculovirus gp64 (30) and Semliki Forest virus E1/E2 fusion protein (20), but they are appreciably larger than the 3-nm (initial conductance, ⬃0.5 nS) pores induced by influenza virus HA (23, 39, 40, 46). A larger pore size may indicate that more Env trimers than HA trimers participate in pore formation. Unlike fusion pores formed by influenza virus HA, pores induced by Env protein did not flicker. Rather, they remained open and steadily enlarged. Thus, although HA and Env of Mo-MuLV have similar core structures, as determined

FIG. 2. Relative fusion activities of Env constructs monitored by a three-fluorescent-dye assay. Effector (293T) cells expressing various Env constructs (as indicated in labels to the left) were labeled with CaAM (green fluorescence). Target 3T3 (left panels) and XC6 (right panels) cells were colabeled with CMAC and PKH26 that have blue and red fluorescence, respectively. Cells were allowed to form contacts for 1 h at 4°C and then were incubated at 37°C for either 60 min (3T3 target cells) or 10 min (XC6 target cells). Fluorescence images for each dye were acquired, pseudocolored, and superimposed on each other. The resulting colors are as follows: green for effector cells, purplish for double-labeled target cells, and white or near white for the fused cells (shown by arrows). The occasional white spots seen for 632* and 595* resulted from a spatial overlap between unfused effector and target cells that occurs when part of one cell lays on the top of another.

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tween cells just brought into contact (Fig. 4B). This suggests that for these cells binding was fast compared to fusion itself. This conclusion assumes that Env-receptor binding was sufficiently insensitive to temperature that it was complete after the 1-h incubation at 4°C and that the time delay was small between pore formation (measured electrically) and pore enlargement (measured by permeation of calcein). It is likely that this delay was small because calcein should have been able to pass through the relatively large initial fusion pore and, in addition, the pore readily enlarged (Fig. 6). Residues 602 to 616 are not required for membrane fusion. For some viral Env proteins, such as for Env of human immunodeficiency virus type 1, syncytia may not form despite the occurrence of fusion (13). We have now shown that the previously observed blockage of appearance of syncytia by R peptide (the C-terminal 16 amino acid residues 617 to 632) of Mo-MuLV (18, 35, 36) is due to prevention of fusion pore formation: deletion of the R peptide greatly promotes MoMuLV Env-mediated fusion. The presence of an R peptide can inhibit fusion induced by other viral Env proteins as well, but this is not universally the case. A chimera between the ectodomain and the TM domain of simian immunodeficiency virus

FIG. 3. Extent of fusion between 293T cells expressing Env protein and either 3T3 fibroblasts (A) and XC6 cells (B). Fusion between effector and target cells was quantified based on fluorescent dye redistribution. (A) After the prebinding of effector and 3T3 cells at 4°C, cells were incubated at 37°C for 1 h, and fusion was assessed by determining the fraction of cell pairs stained with all three fluorescent probes. (B) Extent and the time dependence of Env-induced fusion to XC6 cells. Effector cells were prebound to target cells at 4°C, and the temperature was subsequently raised to 37°C for either 10 min (striped bars) or 40 min (open bars). Error bars represent the standard error of the mean.

crystallographically (4, 15), the properties of their fusion pores are different. Perhaps the lack of flickering for pores formed by Env of Mo-MuLV, by baculovirus gp64 protein, and by Semliki Forest virus E1/E2 protein is related to their larger initial size. Binding is not a rate-limiting step in Mo-MuLV Env fusion. It had been known that the processed Mo-MuLV Env protein (Env 616*) promoted syncytium formation more effectively with the XC6 cell line than with murine 3T3 fibroblasts and that Env 632* caused syncytium formation with XC cells but not with 3T3 cells (18, 21). We have now demonstrated that fusion pores themselves occur to a greater extent and much more quickly with XC6 cells as target and that there was significant pore formation between Env 632*-expressing cells and XC6 cells (Fig. 3B) but not to 3T3 fibroblasts (Fig. 3A). These correlations indicate that the previously observed differences in syncytium formation occur at the point of membrane fusion rather than at the subsequent steps required for syncytia to form. The kinetics for redistribution of cytoplasmic marker between 293T and XC6 cells that had been prebound at 4°C (Fig. 4A) were similar to the kinetics for fusion pores to form be-

FIG. 4. Kinetics of Env-mediated fusion pore formation as measured by the onset of calcein redistribution (A) and electrical measurements of fusion pores (B). (A) Lag times from a temperature jump from 4 to 37°C (time ⫽ 0) until the onset of calcein transfer into a target cell were plotted as cumulative distributions. Env 632* and 595* Env proteins failed to form a pore 10 min after the temperature was raised. Env 616* induced fusion pores after a shorter delay than MelR⫺, which, in turn, induced fusion with less of a delay than did Env 601*. (B) Lag times were measured electrically (at 37°C) as the time intervals between establishing physical contact (by manipulating an effector cell into proximity of a target cell) and fusion pore formation (detected by capacitance measurements). The lag times measured electrically for Env 601* (䊐) and 616* (E) were similar to their lag times measured by dye spread measurements (see panel A). Only cells that fused were used to obtain the cumulative distributions in panels A and B. Thus, the fraction fused in these distributions plateau at long times after acidification to the value of 1.

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FIG. 5. Representative traces of fusion pores formed between 293T expressing Env 616*, Env 601*, and Env MelR⫺ and XC6 cells. All experiments were carried out at 37°C. After the patch clamping of an effector cell, a whole-cell configuration was established, and the cell was lifted from its supporting coverglass and brought in contact with a solitary XC6 cell. Fusion pore formation was detected shortly thereafter by characteristic changes in the electrically recorded cell admittance. Fusion pore conductance was calculated off-line. (See Materials and Methods for details.)

gp160 protein and the full-length CT of Mo-MuLV Env protein did not promote fusion, whereas fusion occurred for the chimera lacking the R peptide (but containing the remaining 16 residues of the CT of Mo-MuLV Env) (45). In contrast, a chimera between the ectodomain of human T-cell leukemia virus type I and the TM domain and full-length CT (which contains the R peptide) from Friend murine leukemia virus supported fusion (12). In the absence of the R peptide, the CT (defined, according to hydrophobicity, as residues 601 and greater) does not strongly affect pore formation. The lower expression levels for Env 601* could account for its slower kinetics and lower extents of fusion compared to Env 616*. In the case of influenza virus HA, relatively small increases in the density of HA greatly decrease delay times from triggering fusion by acidification until pore formation and significantly increase the extent of fusion (14, 24). The S-shape of the cumulative distributions for fusion kinetics (Fig. 5A) indicates that fusion is a multistep process with multiple Env trimers required to act in concert to form a pore (3, 11, 30). The longer delays before commencement of the S-shape’s rising phase for Env 601* is consistent with a lower density of fusion protein. After pore formation, the CT (residues 601 to 616) does not appear to have any influence on Env-induced pores: the initial conductances and enlargements of fusion pores were similar for Env 601*, Env 616*, and Env MelR⫺ (Fig. 5 and 6A). Possible roles of the CT in controlling Env-mediated membrane fusion. Hemifusion is the merger of contacting leaflets of two membranes, while distal, inner leaflets remain distinct and form a bilayer known as a hemifusion diaphragm that continues to separate aqueous compartments. Hemifusion is thought to be a key intermediate of fusion. As evidence for this view, a number of mutations of fusion proteins from several different viruses have been shown to result in lipid dye spread without mixing of aqueous dye when the mutant proteins were expressed on cell surfaces (1, 6–8, 19, 22, 25, 26, 31). But no mutant of Mo-MuLV Env created so far has been shown to produce hemifusion. If viral fusion proteins, in general, induce hemifusion as an intermediate, it is likely that Mo-MuLV Env


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does so as well. Assuming this is the case, how may the CT of Mo-MuLV Env be involved in regulating fusion? One possibility would be that residues 595 to 616 form an amphiphilic ␣-helix that interacts with and destabilizes the hemifusion diaphragm, but the R peptide prevents the interaction (Rozenberg et al., submitted). However, residues 602 to 616 are not critical for fusion. On the other hand, Env MelR⫺, which contains residues 1 to 599 of Mo-MuLV followed by an amphiphilic helix (Rozenberg et al., submitted), is fully supportive of fusion. Since Env 598* did not generate syncytia (Rozenberg et al., submitted), it may not have induced fusion. It remains possible that Env 601* and Env MelR⫺ contain a C-terminal amphiphilic helix required for Mo-MuLV Env-mediated fusion but that this helix is lost in Env 598*. The deletion of the charged arginine (position 601), per se, from Env 601* is probably not the reason Env 598* does not promote syncytia: the addition of serine and arginine to Env 595* (Env 595SR) does not lead to syncytium formation (Rozenberg et al., submitted). Alternatively, since residues 602 to 616 are not required for fusion activity, the membrane-proximal, CT region may not directly create fusion pores. But truncations, point mutations,

FIG. 6. Formation and enlargement of fusion pores formed by Env constructs. Fusion pores induced between Env-expressing 293T cells and XC6 cells were detected in a whole-cell patch-clamp mode by electrical capacitance measurements, and the average pore conductance profiles were constructed. (A) Average initial pores formed by Env 616*, Env 601*, and Env MelR⫺. The error bars are not shown for MelR⫺ for visual clarity. (B) Enlargement of an average fusion pore within the first 100 s of opening. In both panels A and B, Env 616*-induced pore conductances (n ⫽ 17) are shown by solid circles, those formed by Env 601* (n ⫽ 10) are shown by open circles, and those formed by Env MelR⫺ (n ⫽ 11) are shown by triangles. Error bars are the standard errors of the mean. The greater average pore conductance and unusually large error bars for Env 601* were caused by fusion pores that enlarged very quickly in 2 of the 10 experiments. When these two experiments were disregarded, the initial conductance profiles and subsequent enlargement were identical for Env 601* and Env 616* (not shown).

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and deletions of residues within this stretch can strongly influence the fusion activity of Env protein and, in some cases, circumvent the R-peptide block (18). Thus, while the CT would not be required for fusion, the precise amino acid sequence of the CT (perhaps through its secondary structure) that is present may affect the ability of other regions of Env to cause fusion. A similar phenomenology occurs in the case of influenza virus HA. The CT of HA is also not required for fusion, but altering it can strongly affect fusion (23, 27). A CT can affect the conformation of an ectodomain: the ectodomain of Env SIV239 is altered by truncation of the CT (38). As proposed for influenza HA (22, 25), it may be that the CT of Mo-MuLV Env indirectly regulates fusion by affecting the ectodomain and or the MS domain of Env at some stage during the fusion process. The CT would not be required but its presence would control whether the ectodomain could cause hemifusion and whether the MS domain (in cooperation with the ectodomain) could create pores. That is, the ectodomain and the MS domain do not function completely independently of the CT (23). The presence of the R peptide may hinder fusion by altering, at some point during the fusion process, the configurations of ectodomains and/or MS domains within individual trimers or the ability of trimers to interact with each other. The findings with Env 595* and Env 632* suggest that ectodomains, MS domains, and CTs do not function independently but rather function synergistically. Env 595* failed to promote hemifusion to XC6 cells, and Env 632* did not induce hemifusion with 3T3 cells; in both cases not even lipid dye mixing was observed. Nor did the addition of CPZ induce lipid or aqueous dye spread. In contrast, CPZ promotes aqueous dye spread between HA-expressing cells and erythrocytes when the membranes have hemifused (5, 20). Thus, if hemifusion is an intermediate of full fusion, Env 595* and Env 632* are defective at steps upstream of hemifusion. Because the ectodomains of fusion proteins face outer leaflets, it would be natural to expect that ectodomains cause the merger of outer leaflets that characterizes hemifusion, as observed for influenza virus HA (19, 25). Thus, the absence of hemifusion indicates that altering the membrane-proximal region—either by not processing the CT to remove the R peptide in the case of Env 632* or by deleting the CT and shortening the MS domain in the case of Env 595*—can affect the ability of the ectodomain to cause hemifusion. How the domains of Env interact with each other and how the R peptide affects these interactions depend on molecular features that are not yet appreciated. ACKNOWLEDGMENTS We thank Boris Deriy for writing the software used for analyzing the video images, Gregory Spear and Jocelyn Jakubik for guidance and help in using their fluorescence-activated cell sorting facility, and David Sanders for critically reading the manuscript. This work was supported by National Institutes of Health grants GM27367 (F.S.C.) and GM54787 (G.B.M.) and by Genetic Therapy, Inc./Novartis (Gaithersburg, Md.). REFERENCES 1. Bagai, S., and R. A. Lamb. 1996. Truncation of the COOH-terminal region of the paramyxovirus SV5 fusion protein leads to hemifusion but not complete fusion. J. Cell Biol. 135:73–84. 2. Baker, K. A., R. E. Dutch, R. A. Lamb, and T. S. Jardetzky. 1999. Structural basis for paramyxovirus-mediated membrane fusion. Mol. Cell. 3:309–319. 3. Blumenthal, R., D. P. Sarkar, S. Durell, D. E. Howard, and S. J. Morris. 1996. Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events. J. Cell Biol. 135:63–71. 4. Bullough, P. A., F. M. Hughson, J. J. Skehel, and D. C. Wiley. 1994. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371: 37–43. 5. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of

J. VIROL. gp41 from the HIV envelope glycoprotein. Cell 89:263–273. 6. Chernomordik, L. V., V. A. Frolov, E. Leikina, P. Bronk, and J. Zimmerberg. 1998. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J. Cell Biol. 140:1369–1382. 7. Chernomordik, L. V., E. Leikina, V. Frolov, P. Bronk, and J. Zimmerberg. 1997. An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J. Cell Biol. 136:81–93. 8. Cleverley, D. Z., and J. Lenard. 1998. The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein. Proc. Natl. Acad. Sci. USA 95:3425–3430. 9. Cohen, F. S., and G. B. Melikyan. 1998. Methodologies in the study of cell-cell fusion. Methods 16:215–226. 10. Crawford, S., and S. P. Goff. 1985. A deletion mutation in the 5⬘ part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. J. Virol. 53:899–907. 11. Danieli, T., S. L. Pelletier, Y. I. Henis, and J. M. White. 1996. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J. Cell Biol. 133:559–569. 12. Denesvre, C., P. Sonigo, A. Corbin, H. Ellerbrok, and M. Sitbon. 1995. Influence of transmembrane domains on the fusogenic abilities of human and murine leukemia retrovirus envelopes. J. Virol. 69:4149–4157. 13. Dimitrov, D. S., H. Golding, and R. Blumenthal. 1991. Initial stages of HIV-1 envelope glycoprotein-mediated cell fusion monitored by a new assay based on redistribution of fluorescent dyes. AIDS Res. Hum. Retroviruses 7:799– 805. 14. Ellens, H., J. Bentz, D. Mason, F. Zhang, and J. M. White. 1990. Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density. Biochemistry 29:9697–9707. 15. Fass, D., S. C. Harrison, and P. S. Kim. 1996. Retrovirus envelope domain at 1.7 angstrom resolution. Nat. Struct. Biol. 3:465–469. 16. Green, N., T. M. Shinnick, O. Witte, A. Ponticelli, J. G. Sutcliffe, and R. A. Lerner. 1981. Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide. Proc. Natl. Acad. Sci. USA 78:6023–6027. 17. Hernandez, L. D., L. R. Hoffman, T. G. Wolfsberg, and J. M. White. 1996. Virus-cell and cell-cell fusion. Annu. Rev. Cell. Dev. Biol. 12:627–661. 18. Januszeski, M. M., P. M. Cannon, D. Chen, Y. Rozenberg, and W. F. Anderson. 1997. Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J. Virol. 71:3613–3619. 19. Kemble, G. W., T. Danieli, and J. M. White. 1994. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell 76:383–391. 20. Lanzrein, M., N. K. Esermann, R. Weingart, and C. Kempf. 1993. Early events of Semliki Forest virus-induced cell-cell fusion. Virology 196:541–547. 21. McClure, M. O., M. A. Sommerfelt, M. Marsh, and R. A. Weiss. 1990. The pH independence of mammalian retrovirus infection. J. Gen. Virol. 71: 767–773. 22. Melikyan, G. B., S. A. Brener, D. C. Ok, and F. S. Cohen. 1997. Inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion. J. Cell Biol. 136:995–1005. 23. Melikyan, G. B., S. Lin, M. G. Roth, and F. S. Cohen. 1999. Amino acid sequence requirements of the transmembrane and cytoplasmic domains of influenza virus hemagglutinin for viable membrane fusion. Mol. Biol. Cell. 10:1821–1836. 24. Melikyan, G. B., W. D. Niles, and F. S. Cohen. 1995. The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface. J. Gen. Physiol. 106:783–802. 25. Melikyan, G. B., J. M. White, and F. S. Cohen. 1995. GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes. J. Cell Biol. 131:679–691. 26. Munoz-Barroso, I., S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal. 1998. Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41. J. Cell Biol. 140:315–323. 27. Ohuchi, M., C. Fischer, R. Ohuchi, A. Herwig, and H. D. Klenk. 1998. Elongation of the cytoplasmic tail interferes with the fusion activity of influenza virus hemagglutinin. J. Virol. 72:3554–3559. 28. Pinter, A., T. E. Chen, A. Lowy, N. G. Cortez, and S. Silagi. 1986. Ecotropic murine leukemia virus-induced fusion of murine cells. J. Virol. 57:1048– 1054. 29. Pinter, A., and W. J. Honnen. 1983. Topography of murine leukemia virus envelope proteins: characterization of transmembrane components. J. Virol. 46:1056–1060. 30. Plonsky, I., and J. Zimmerberg. 1996. The initial fusion pore induced by baculovirus GP64 is large and forms quickly. J. Cell Biol. 135:1831–1839. 31. Qiao, H., R. T. Armstrong, G. B. Melikyan, F. S. Cohen, and J. M. White. 1999. A specific point mutant at position 1 of the influenza hemagglutinin fusion peptide displays a hemifusion phenotype. Mol. Biol. Cell 10:2759– 2769. 32. Ragheb, J. A., and W. F. Anderson. 1994. pH-independent murine leukemia

VOL. 74, 2000

33. 34. 35.

36. 37.

38.

39.

virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68:3220–3231. Ragheb, J. A., and W. F. Anderson. 1994. Uncoupled expression of Moloney murine leukemia virus envelope polypeptides SU and TM: a functional analysis of the role of TM domains in viral entry. J. Virol. 68:3207–3219. Ratinov, V., I. Plonsky, and J. Zimmerberg. 1998. Fusion pore conductance: experimental approaches and theoretical algorithms. Biophys. J. 74:2374– 2387. Rein, A., J. Mirro, J. G. Haynes, S. M. Ernst, and K. Nagashima. 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68:1773–1781. Schultz, A., and A. Rein. 1985. Maturation of murine leukemia virus env proteins in the absence of other viral proteins. Virology 145:335–339. Soneoka, Y., P. M. Cannon, E. E. Ramsdale, J. C. Griffiths, G. Romano, S. M. Kingsman, and A. J. Kingsman. 1995. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23:628–633. Spies, C. P., G. D. Ritter, Jr., M. J. Mulligan, and R. W. Compans. 1994. Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain. J. Virol. 68:585–591. Spruce, A. E., A. Iwata, J. M. White, and W. Almers. 1989. Patch clamp


40. 41. 42. 43. 44. 45. 46.

455

studies of single cell-fusion events mediated by a viral fusion protein. Nature 342:555–558. Tse, F. W., A. Iwata, and W. Almers. 1993. Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion. J. Cell Biol. 121:543–552. Weiss, R. A. 1993. Cellular receptors and viral glycoproteins involved in retrovirus entry, p. 1–108. In J. A. Levy (ed.), The Retroviridae, vol. 2. Plenum Press, New York, N.Y. Weissenhorn, W., A. Carfi, K. H. Lee, J. J. Skehel, and D. C. Wiley. 1998. Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol. Cell 2:605–616. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387: 426–430. Witte, O. N., A. Tsukamoto-Adey, and I. L. Weissman. 1977. Cellular maturation of oncornavirus glycoproteins: topological arrangement of precursor and product forms in cellular membranes. Virology 76:539–553. Yang, C., and R. W. Compans. 1996. Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide. J. Virol. 70:248–254. Zimmerberg, J., R. Blumenthal, D. P. Sarkar, M. Curran, and S. J. Morris. 1994. Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion. J. Cell Biol. 127:1885–1894.