Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.     

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.     

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.     

Morphological Aspects of the Uptake of Simian Virus 40 by Permissive Cells

After exposure of permissive cells to simian virus 40 (SV40), single particles were engulfed by the cell membrane and transported to the nucleus. The cell membrane closed tightly around the particles, increasing their diameter from 40 to 55 nm. The cell membrane was lost during interaction with the nuclear membranes, and particles of the original size were found in the nucleus 1 hr after infection. Uncoating of these nuclear particles occurred rapidly, and none could be found 4 hr after infection. Viral progeny appeared 24 hr after infection.     

The Simian virus 40 late viral protein VP4 disrupts the nuclear envelope for viral release

Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release.     

The SV40 late protein VP4 is a viroporin that forms pores to disrupt membranes for viral release

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ～3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release.     

Replication of Epstein-Barr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells.

We have studied by means of electron microscopy and immunofluorescence the different steps of the replication of the P3HR1 strain of Epstein-Barr virus in Raji cells. The virus entered the cell by fusion of the viral envelope with the plasma membrane, followed by the disintegration of the capsid. In some cases, the migration of nucleocapsids toward the nuclear membrane was observed. The synthesis of new virions began as early as 7 h after infection (in the case of a high multiplicity of infection [MOI]-800 particles per cell) and took place in low-electron-density areas of the nucleus. A viral envelope was acquired by budding either through the nuclear membrane or more often through membranes of the Golgi apparatus or cytoplasmic vacuoles. Comparing immunofluorescence and electron microscopic data a good correlation was found between the presence of early antigen and ultrastructurally altered cells, as well as between the presence of viral capsid antigen and virus-producing cells. With different MOIs, different types of viral cycles were observed: at a low MOI (less than or equal to 50 particles per cell), a nonproducer cycle was induced, with early antigen synthesis only; at a higher MOI (100 particles per cell), a transient production of a small amount of virions was observed, and at a high MOI (greater than or equal to 300 particles per cell), a productive cycle was the rule.     

Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P₂) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.     

Fusion of SV40-induced endocytotic vacuoles with the nuclear membrane

The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.     

Fusion of Sendai Viruses or Subviral Envelope Components with Chicken Erythrocytes Observed by Freeze-Fracture Electron Microscopy

Four different types of envelope of Sendai virus or subviral components, that is, infectious and non-infectious virions, reassembled envelope particles (REP), and Tween-ether-treated envelope fragments (TE), were studied comparatively for membrane interactions with chicken erythrocytes by freeze-fracture electron microscopy, specifically for membrane alteration by envelope fusion. The freeze-fracture replicas of the attachment of the four envelopes in the cold exhibited a common pattern of impressions with attached envelopes, although the fracture plane traversed from erythrocyte to envelope at the periphery of the contact areas of three of the envelopes but not of TE, where the fracture plane mostly cut only through erythrocyte membranes impressed with TE. The freeze-fracture replicas of the four envelopes reacting with erythrocytes after a short incubation period at 37 C exhibited distinctive features: infectious virions and REP displayed evidence of envelope fusion, but non-infectious virions and TE showed a particular pattern of envelope association without fusion. Our data demonstrate that the pattern specific for envelope fusion is the formation of a continuous membrane from envelope to cell membrane in a cross fracture of an erythrocyte.     

N-ethylmaleimide-sensitive factor is associated with the nuclear envelope

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg(2+)-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg(2+)-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.     

The movement and invasion of an insect baculovirus in tracheal cells of the armyworm, Pseudaletia unipuncta

The hypertrophy nuclear polyhedrosis virus of the armyworm, Pseudaletia unipuncta, causes a unique gradient of infected cells to form on the trachea. The movement and invasion of the virus apparently were not through adjacent intercellular membranes. The enveloped viruses emerged from the initially infected cell into an area between the cell plasma membrane and basal lamina, and then entered the uninfected tracheal cell either by lateral attachment and fusion of the viral envelope and the plasma membrane or by viropexis. The two methods of viral invasion into the cell suggest the presence of at least two phenotypically different enveloped viruses. Viropexis was initiated with an alignment of the peplomer spikes with regularly spaced, short radial striations on the inner coat of the plasma membrane. At a late state in viropexis, the viral envelope fused with the vacuole membrane, and an opening developed below the site of membrane fusion through which the nucleocapsid might enter the cytoplasm. Some nucleocapsids in membrane-lined vesicles resulting from viropexis appeared to be in a state of dissolution. Naked nucleocapsids were found along the nuclear envelope and within the nucleoplasm. No uncoating of the nucleocapsids was observed at the nucleopores, but uncoating seemed to occur in the nucleoplasm. Nucleocapsids were also found in the cytoplasm of nonsusceptible fat body cells, in which virus replication was not observed.     

The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

Key role of polyphosphoinositides in dynamics of fusogenic nuclear membrane vesicles

The role of phosphoinositides has been thoroughly described in many signalling and membrane trafficking events but their function as modulators of membrane structure and dynamics in membrane fusion has not been investigated. We have reconstructed models that mimic the composition of nuclear envelope precursor membranes with naturally elevated amounts of phosphoinositides. These fusogenic membranes (membrane vesicle 1(MV1) and nuclear envelope remnants (NER) are critical for the assembly of the nuclear envelope. Phospholipids, cholesterol, and polyphosphoinositides, with polyunsaturated fatty acid chains that were identified in the natural nuclear membranes by lipid mass spectrometry, have been used to reconstruct complex model membranes mimicking nuclear envelope precursor membranes. Structural and dynamic events occurring in the membrane core and at the membrane surface were monitored by solid-state deuterium and phosphorus NMR. "MV1-like" (PC∶PI∶PIP∶PIP(2), 30∶20∶18∶12, mol%) membranes that exhibited high levels of PtdIns, PtdInsP and PtdInsP(2) had an unusually fluid membrane core (up to 20% increase, compared to membranes with low amounts of phosphoinositides to mimic the endoplasmic reticulum). "NER-like" (PC∶CH∶PI∶PIP∶PIP(2), 28∶42∶16∶7∶7, mol%) membranes containing high amounts of both cholesterol and phosphoinositides exhibited liquid-ordered phase properties, but with markedly lower rigidity (10-15% decrease). Phosphoinositides are the first lipids reported to counterbalance the ordering effect of cholesterol. At the membrane surface, phosphoinositides control the orientation dynamics of other lipids in the model membranes, while remaining unchanged themselves. This is an important finding as it provides unprecedented mechanistic insight into the role of phosphoinositides in membrane dynamics. Biological implications of our findings and a model describing the roles of fusogenic membrane vesicles are proposed.     

Herpes simplex virus 1 envelopment follows two diverse pathways

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.     

Role of nuclear pore complex in simian virus 40 nuclear targeting.

Cytoplasmically injected simian virus 40 (SV40) virions enter the nucleus through nuclear pore complexes (NPCs) and can express large T antigen shortly thereafter (J. Clever, M. Yamada, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 88:7333-7337, 1991). The nuclear import of the protein components of introduced SV40 was reversibly arrested by chilling and energy depletion, corroborating our previous observation that the nuclear entry of injected SV40 is blocked in the presence of wheat germ agglutinin and an antinucleoporin monoclonal antibody (mAb414), general inhibitors of NPC-mediated import. The nuclear accumulation of virion protein components and large T antigen in nonpermissive NIH 3T3 cells was similar to that in the permissive host, indicating that the ability to use NPCs as a route of nuclear entry appears to be a general property of the injected virus. Injected virions were capable of completing their lytic cycle and forming plaques in permissive cells. During the early phase of SV40 infection, the cytoplasmic injection of mAb414 effectively blocked nuclear T-antigen accumulation for up to 8 h of infection but had very little effect after 12 h of infection. The time-dependent interference with nuclear T-antigen accumulation by the antinucleoporin antibody is consistent with the hypothesis that the infecting virions enter the nucleus through NPCs. The interference study also suggests that the early phase of infection consists of at least two steps: a step for virion cell entry and intracytoplasmic trafficking and a step for virion nuclear entry followed by large-T-antigen gene expression and subsequent nuclear localization of the gene product. Virions were visualized as electron-dense particles in ultrathin sections of samples in which transport was permitted or arrested. In the former cells, electron-dense particles were predominantly observed in the nucleus. The virions were distributed randomly and nonuniformly in the nucleoplasm but were not observed in heterochromatin or in nucleoli. In the latter cells, the electron-dense particles were seen intersecting the nuclear envelope, near the inner nuclear membrane, and in NPCs. In tangential cross sections of NPCs, which appeared as donut-shaped structures, a spherical electron-dense particle was observed in the center of the structure. Immunoelectron microscopy revealed that NPCs were selectively decorated with 5-nm colloidal gold particles-anti-Vp1 immunoglobulin G at the cytoplasmic entrance to and in NPCs, confirming that the morphologically observed electron-dense particles in NPCs contain the viral structural protein. These results support the hypothesis that the nuclear import of SV40 is catalyzed through NPCs by an active transport mechanism that is similar to that of other karyophiles.     

Egress of alphaherpesviruses: comparative ultrastructural study

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.     

Sendai virus envelopes can mediate Epstein-Barr virus binding to and penetration into Epstein-Barr virus receptor-negative cells

Epstein-Barr virus (EBV) receptor-negative cells were treated with UV-inactivated Sendai virus (SV) or with reconstituted SV envelopes having a low hemolytic activity and then assayed for EBV binding or for susceptibility to EBV infection. EBV binding was assessed by using both unlabeled and fluoresceinated EBV preparations. It was found that SV or SV envelope treatment renders these cells able to bind EBV. Various experiments were performed to clarify the mechanism of this SV-induced binding. The EBV receptor-negative 1301 cells were treated with SV either at 0°C or at both 0 and 37°C successively and then examined for EBV binding at 0°C. It was thus found that when SV treatment was performed exclusively at 0°C, the target cells showed higher fluorescence intensity after their incubation with fluoresceinated EBV. In addition, Clostridium perfringens neuraminidase treatment of 1301 cells did not induce any EBV binding to these cells. These data indicate that EBV binding is not due to the disturbance of the cell membrane by SV envelope fusion or to the uncovering of EBV binding sites on the cells after the enzymatic action of SV neuraminidase. Moreover, bound EBV was partly eluted from SV-treated 1301 cells at 37°C, and the treatment of EBV with C. perfringens neuraminidase inhibited its SV-mediated binding. These data indicate that EBV binds to the hemagglutinin-neuraminidase of SV on the target cell surface and that a fraction of the bound EBV becomes irreversibly associated with the SV-treated cell membrane. Our data also show that EBV can penetrate into 1301 cells which have incorporated SV envelopes into their membrane, as demonstrated by the induction of the EBV-determined nuclear antigen by B95-8 EBV in SV envelope-treated 1301 cells.     

Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze-fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed.