The ESCRT-I subunit TSG101 controls endosome-to-cytosol release of viral RNA

Like other enveloped viruses, vesicular stomatitis virus infects cells through endosomes. There, the viral envelope undergoes fusion with endosomal membranes, thereby releasing the nucleocapsid into the cytoplasm and allowing infection to proceed. Previously, we reported that the viral envelope fuses preferentially with the membrane of vesicles present within multivesicular endosomes. Then, these intra-endosomal vesicles (containing nucleocapsids) are transported to late endosomes, where back-fusion with the endosome limiting membrane delivers the nucleocapsid into the cytoplasm. In this study, we show that the tumor susceptibility gene 101 (Tsg101) subunit of the endosomal sorting complexes required for transport (ESCRT)-I complex, which mediates receptor sorting into multivesicular endosomes, is dispensable for viral envelope fusion with endosomal membranes and viral RNA transport to late endosomes but is necessary for infection. Our data indicate that Tsg101, in contrast to the ESCRT-0 component Hrs, plays a direct role in nucleocapsid release from within multivesicular endosomes to the cytoplasm, presumably by controlling the back-fusion process. We conclude that Tsg101, through selective interactions with its partners including Hrs and Alix, may link receptor sorting and lysosome targeting to the back-fusion process involved in viral capsid release.     

Endosome-to-cytosol transport of viral nucleocapsids

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors.     

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.     

Promotion of vesicular stomatitis virus fusion by the endosome-specific phospholipid bis(monoacylglycero)phosphate (BMP)

Vesicular stomatitis virus (VSV) is a prototypic virus commonly used in studies of endocytosis and membrane trafficking. One proposed mechanism for VSV entry involves initial fusion with internal vesicles of multivesicular endosomes followed by back-fusion of these vesicles into the cytoplasm. One feature of endosomal internal vesicles is that they contain the lipid bis(monoacylglycero)phosphate (BMP). Here, we show that the presence of BMP significantly increases the rate of VSV G-mediated membrane fusion. The increased fusion was selective for VSV and was not evident for another enveloped virus, influenza virus. Our data provide a biological rationale for a two-step infection reaction during VSV entry, and suggest that BMP preferentially affects the ability of VSV G to mediate lipid mixing during membrane fusion.     

Vaccinia virus entry into cells via a low-pH-dependent endosomal pathway

Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.     

Trans-infection but Not Infection from within Endosomal Compartments after Cell-to-cell HIV-1 Transfer to CD4+ T Cells

Cellular contacts between HIV-1-infected donor cells and uninfected primary CD4(+) T lymphocytes lead to virus transfer into endosomes. Recent evidence suggests that HIV particles may fuse with endosomal membranes to initiate a productive infection. To explore the role of endocytosis in the entry and replication of HIV, we evaluated the infectivity of transferred HIV particles in a cell-to-cell culture model of virus transmission. Endocytosed virus led to productive infection of cells, except when cells were cultured in the presence of the anti-gp120 mAb IgGb12, an agent that blocks virus attachment to CD4, suggesting that endocytosed virus was recycled to the outer cell surface. Confocal microscopy confirmed the colocalization of internalized virus antigen and the endosomal marker dynamin. Additionally, virus transfer, fusion, or productive infection was not blocked by dynasore, dynamin-dependent endosome-scission inhibitor, at subtoxic concentrations, suggesting that the early capture of virus into intracellular compartments did not depend on endosomal maturation. Our results suggest that endocytosis is not a mechanism of infection of primary CD4 T cells, but may serve as a reservoir capable of inducing trans-infection of cells after the release of HIV particles to the extracellular environment.     

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.     

Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.     

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.     

New insights into the role of endosomal proteins for African swine fever virus infection

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.     

Two distinct low-pH steps promote entry of vaccinia virus

Entry of vaccinia virus into cells occurs by an endosomal route as well as through the plasma membrane. Evidence for an endosomal pathway was based on findings that treatment at a pH of <6 of mature virions attached to the plasma membrane enhances entry, whereas inhibitors of endosomal acidification reduce entry. Inactivation of infectivity by low-pH treatment of virions prior to membrane attachment is characteristic of many viruses that use the endosomal route. Nevertheless, we show here that the exposure of unattached vaccinia virus virions to low pH at 37 degrees C did not alter their infectivity. Instead, such treatment stably activated virions as indicated by their accelerated entry upon subsequent addition to cells, as measured by reporter gene expression. Moreover, the rate of entry was not further enhanced by a second low-pH treatment following adsorption to the plasma membrane. However, the entry of virions activated prior to adsorption remained sensitive to inhibitors of endosomal acidification, whereas virions treated with low pH after adsorption were resistant. Activation of virions by low pH was closely mimicked by proteinase digestion, suggesting that the two treatments operate through a related mechanism. Although proteinase cleavage of the virion surface proteins D8 and A27 correlated with activation, mutant viruses constructed by individually deleting these genes did not exhibit an activated phenotype. We propose a two-step model of vaccinia virus entry through endosomes, in which activating or unmasking the fusion complex by low pH or by proteinase is rate limiting but does not eliminate a second low-pH step mediating membrane fusion.     

Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.     

The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.     

Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent

Due to the limited ultrastructural information about the coronavirion, little is known about the interactions acting at the interface between nucleocapsid and viral envelope. Knowing that subtle mutations in the carboxy-terminal endodomain of the M protein are already lethal, we have now probed the equivalent domain of the spike (S) protein by extending it terminally with a foreign sequence of 27 kDa: the green fluorescent protein (GFP). When expressed individually in murine cells, the S-GFP chimeric protein induced the formation of fluorescent syncytia, indicating that it was synthesized and folded properly, trimerized, and transported to the plasma membrane, where it exhibited the two key S protein functions, i.e., interaction with virus receptor molecules and membrane fusion. Incorporation into virus-like particles demonstrated the assembly competence of the chimeric spike protein. The wild-type S gene of mouse hepatitis coronavirus (MHV) was subsequently replaced by the chimeric construct through targeted recombination. A viable MHV-SGFP was obtained, infection by which could be visualized by the fluorescence induced. The efficiency of incorporation of the chimeric protein into particles was, however, reduced relative to that in wild-type particles which may explain, at least in part, the reduced infectivity produced by MHV-SGFP infection. We conclude that the incorporation of spikes carrying the large GFP moiety is apparently impaired by geometrical constraints and selected against during the assembly of virions. Probably due to this disadvantage, deletion mutants, having lost the foreign sequences, rapidly evolved and outcompeted the chimeric viruses during virus propagation. The fluorescent MHV-SGFP will now be a convenient tool to study coronaviral cell entry.     

Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice.

Transgenic mice that express the hepatitis B virus core protein were used to examine factors that influence the intracellular localization of nucleocapsid particles in the primary hepatocyte in vivo. In this model, viral nucleocapsid particles are strictly localized to the nucleus of the hepatocyte except when the nuclear membrane dissolves during cell division, at which time they enter the cytoplasm. The cytoplasmic nucleocapsid particles do not reenter the nucleus, however, when the nuclear membrane re-forms after cell division. The data support the notion that nucleocapsid particles can form de novo within the nucleus, and they suggest that performed nucleocapsid particles cannot be transported across the intact nuclear membrane in either direction. The results imply that nucleocapsid disassembly is probably required for entry of the hepadnaviral genome into the nucleus, and they question the role of the intranuclear viral nucleocapsid particle during the viral life cycle.     

Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.     

Endocytic entry of HIV-1

Enveloped viruses enter target cells by membrane fusion or endocytosis. In the latter case, fusion of the viral envelope is induced by the acidic pH of the endocytic vesicle [1]. As with most other retroviruses, entry of the human immunodeficiency virus (HIV) is thought to be exclusively by pH-independent membrane fusion after interaction of its envelope with CD4 and a chemokine co-receptor on the target cell [2,3]. Expression of CD4 on the virus-producing cell impairs the release and infectivity of HIV-1(NL4-3) particles [4-6]. In sharp contrast, we found that the infectivity of another HIV isolate, HIV-1SF2, was enhanced by expression of CD4 on the producer cells, which correlated with significantly increased amounts of viral proteins in the vesicular fraction of target cells. Endocytic inhibitors decreased infectivity of HIV-1SF2 but enhanced that of HIV-1 NL4-3. Expression of CD4 in the producer cell did not remove gp41 from HIV-1SF2 virions. With these cells, the formation of syncytia could be induced by acidic medium. Thus, HIV-1SF2 can enter the cytoplasm by an endocytic route after activation of gp41 by the acidic pH of endocytic vesicles. Endocytic entry might expand the range of cells that HIV could infect and should be considered in antiviral strategies against AIDS.     

Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (<pH 6) equivalent to the pH of late endolysosomal compartments allowed the virus to bypass the endosomal route of infection. Acid exposure of virions in the absence of target membranes triggered the class II-like Gc fusion protein to form extremely stable oligomers that were resistant to SDS and temperature dissociation and concomitantly compromised virus infectivity. By targeted mutagenesis of conserved histidines in Gn and Gc, we demonstrated that mutation of a single histidine (H857) in Gc completely abrogated virus entry, as well as acid-induced Gc oligomerization. In conclusion, our data suggest that after endocytic uptake, RVFV traffics to the acidic late endolysosomal compartments, where histidine protonation drives the reorganization of the Gc fusion protein that leads to membrane fusion.     

Molecular mechanisms of flavivirus membrane fusion

Flaviviruses comprise a number of important human pathogens including yellow fever, dengue, West Nile, Japanese encephalitis and tick-borne encephalitis viruses. They are small enveloped viruses that enter cells by receptor-mediated endocytosis and release their nucleocapsid into the cytoplasm by fusing their membrane with the endosomal membrane. The fusion event is triggered by the acidic pH in the endosome and is mediated by the major envelope protein E. Based on the atomic structures of the pre- and post-fusion conformations of E, a fusion model has been proposed that includes several steps leading from the metastable assembly of E at the virion surface to membrane merger and fusion pore formation trough conversion of E into a stable trimeric post-fusion conformation. Using recombinant subviral particles of tick-borne encephalitis virus as a model, we have defined individual steps of the molecular processes underlying the flavivirus fusion mechanisms. This includes the identification of a conserved histidine as being part of the pH sensor in the fusion protein that responds to the acidic pH and thus initiates the structural transitions driving fusion.     

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.