Polycaryocyte formation mediated by Sindbis virus glycoproteins.

The process of cell fusion mediated by Sindbis virus membrane proteins synthesized after infection was examined. At the times after infection at which virus proteins were detectable on the cell surface, Sindbis virus-infected BHK-21 cells were found to express a fusion function after brief treatment at acid pH. In studies employing wild-type virus and temperature-sensitive mutants and testing drug or protease inhibition of virus production, we made the following observations on Sindbis virus-mediated fusion from within. (i) Fusion requires the synthesis of virus glycoproteins and their transport to the cell surface. (ii) Modification of the cell plasma membrane by polypeptides PE2 and E1 alone is not sufficient for expression of the fusion function. (iii) The proteolytic conversion of plasma membrane-associated PE2 to E2 is not essential for fusion. (iv) Glycosylation of virus plasma membrane proteins is essential for fusion. (v) The lesions of Sindbis virus temperature-sensitive mutants do not affect their ability to fuse cells.     

Subcellular location of the major protein antigens of paramyxoviruses revealed by immunoperoxidase cytochemistry

The major structural proteins of Newcastle disease virus and Sendai virus were localized in infected BHK-21 and MDBK cells by ultrastructural immunoperoxidase cytochemistry using antibodies against the individual viral protein antigens. The intracellular glycoproteins were strictly membrane bound, being localized in the rough endoplasmic reticulum (RER), perinuclear spaces, smooth membrane vesicles, and presumed Golgi apparatus. The nucleocapsid proteins were detected exclusively in membrane free cytosol and accumulated there, forming inclusions. The membrane (M) protein was found both in cytosol and on RER. The viral proteins on RER exhibited a distinct site specificity; the glycoproteins were facing the lumen of RER whereas M protein was present at the outer cytoplasmic surface. All the viral proteins were detectable at the plasma membrane where virus assembly takes place. However, their modes of distribution differed remarkably. The glycoproteins were spread widely over the entire cell surface including the areas of virus budding and those of normal morphology, whereas M protein was localized in restricted areas of the membrane, frequently forming a patch of virus specific membrane. The presence of nucleocapsids was confined to the virus particles budding from the plasma membrane. These results complement and extend the earlier morphological and biochemical data on the assembly or morphogenesis of paramyxoviruses.     

Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis.

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.     

Structural basis for membrane fusion by enveloped viruses.

Enveloped viruses such as HIV-1, influenza virus, and Ebola virus express a surface glycoprotein that mediates both cell attachment and fusion of viral and cellular membranes. The membrane fusion process leads to the release of viral proteins and the RNA genome into the host cell, initiating an infectious cycle. This review focuses on the HIV-1 gp41 membrane fusion protein and discusses the structural similarities of viral membrane fusion proteins from diverse families such as Retroviridae (HIV-1), Orthomyxoviridae (influenza virus), and Filoviridae (Ebola virus). Their structural organization suggests that they have all evolved to use a similar strategy to promote fusion of viral and cellular membranes. This observation led to the proposal of a general model for viral membrane fusion, which will be discussed in detail.     

Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain

The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.     

The ion channels coded by viruses

Pathogenicity and virulence are multifactorial traits, depending on interaction of viruses with susceptible cells and organisms. The ion channels coded by viruses, viroporins, represent only one factor taking part in the cascade of interactions between virus and cell, leading to the entry of virus, replication and to profound changes in membrane permeability. The M2 protein from influenza A virus forms proton-selective, pH-regulated channel involved in regulating vesicular pH, a function important for the correct maturation of HA glycoprotein. The NB glycoprotein of influenza B viruses is an integral membrane protein with an ion channel activity. The CM2 protein of influenza C virus is an integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. The picornavirus 3A protein is involved in cell lysis and shows homology with other lytic proteins. Vpu is an oligomeric integral membrane protein encoded by HIV-1, which forms ion channels. The togavirus 6K protein shows structural similarities with other viroporins.     

Mobilization and aggregation of integral membrane proteins in erythrocytes induced by interaction with influenza virus at acidic pH

Effect of influenza virus on erythrocyte membranes was investigated by electron microscopy and fluorescence photobleaching recovery measurements. The virus induced mobilization of integral proteins in erythrocyte membrane at acidic pH, where it fused with the cell membrane to cause hemolysis and also cell fusions but not at neutral pH. At lower temperatures (e.g., 4 degrees C), the proteins aggregated in the membrane and, consequently, large protein-free lipid bilayer area was produced. At higher temperatures (e.g., 37 degrees C) the protein distribution became randomized. Spectrin meshwork underneath the erythrocyte membrane was also markedly modified by the virus at acidic pH. Diffuse fibril structure was converted into dense spots and the membrane area lacking the fibril structure was produced. Isolated hemagglutinin rosettes also caused mobilization and aggregation of the integral proteins at acidic pH but to smaller extent than that induced by virus. The membrane perturbation detected as the protein mobilization by the action of hemagglutinin was assigned to be the cause for envelope fusion.     

Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions

Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-microg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.     

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.     

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

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

Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release.

Flaviviruses are enveloped viruses which accumulate in cellular vacuoles prior to release. The membrane of cell-associated virus contains the proteins pre-M and E. During release of virus the pre-M protein is cleaved, and only its carboxy-terminal segment remains associated with the virus as M protein. Studies of the association of membrane proteins of intracellular and extracellular particles of West Nile virus show that in cell-associated virus the pre-M and E proteins are present as E+pre-M heterodimers. Cleavage of pre-M during release leads to dissociation of the heterodimers: the amino-terminal region of the pre-M protein is lost from the virus, whereas the proteins M and E remain associated with the viral membrane as separate molecules. The E protein of extracellular virus has a tendency to oligomerize into trimers, and both E-protein monomers and trimers are present on extracellular virions. We have prepared partially purified extracellular virus without loss of viral infectivity. These preparations contain approximately 600 physical particles for each PFU. Since purification of cell-associated virus results in significant loss of PFU, an inactivation of virus may occur during this procedure. Preparations of cell-associated virus contained approximately 40,000 physical particles for each PFU.     

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells expressed an 85,000-dalton protein that was precipitated by antisera directed against feline leukemia virus p12, p15, and p30 proteins. No feline oncornavirus-associated cell membrane antigen reactivity was detected on the surfaces of the transformed murine cells by indirect membrane immunofluorescence techniques. The 85,000-dalton feline sarcoma virus-specific protein was also found in feline cells transformed by transfection. However, these cells also contained env gene products. The results of this study demonstrate that the feline sarcoma virus genome is sufficient to transform murine cells and that expression of the 85,000-dalton gag-x protein is associated with transformation of both murine and feline cells transformed by transfection.     

Virus Maturation by Budding

Enveloped viruses mature by budding at cellular membranes. It has been generally thought that this process is driven by interactions between the viral transmembrane proteins and the internal virion components (core, capsid, or nucleocapsid). This model was particularly applicable to alphaviruses, which require both spike proteins and a nucleocapsid for budding. However, genetic studies have clearly shown that the retrovirus core protein, i.e., the Gag protein, is able to form enveloped particles by itself. Also, budding of negative-strand RNA viruses (rhabdoviruses, orthomyxoviruses, and paramyxoviruses) seems to be accomplished mainly by internal components, most probably the matrix protein, since the spike proteins are not absolutely required for budding of these viruses either. In contrast, budding of coronavirus particles can occur in the absence of the nucleocapsid and appears to require two membrane proteins only. Biochemical and structural data suggest that the proteins, which play a key role in budding, drive this process by forming a three-dimensional (cage-like) protein lattice at the surface of or within the membrane. Similarly, recent electron microscopic studies revealed that the alphavirus spike proteins are also engaged in extensive lateral interactions, forming a dense protein shell at the outer surface of the viral envelope. On the basis of these data, we propose that the budding of enveloped viruses in general is governed by lateral interactions between peripheral or integral membrane proteins. This new concept also provides answers to the question of how viral and cellular membrane proteins are sorted during budding. In addition, it has implications for the mechanism by which the virion is uncoated during virus entry.     

Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network.

During the assembly of vaccinia virus, the intracellular mature virus becomes enwrapped by a cellular cisterna to form the intracellular enveloped virus (IEV), the precursor of the extracellular enveloped virus (EEV). In this study, we have characterized the origin of this wrapping cisterna by electron microscopic immunocytochemistry using lectins, antibodies against endocytic organelles, and recombinant vaccinia viruses expressing proteins which behave as Golgi resident proteins. No labelling for endocytic marker proteins could be detected on the wrapping membrane. However, the wrapping membrane labelled significantly for a trans Golgi network (TGN) marker protein. The recycling pathway from endosomes to the TGN appears to be greatly increased following vaccinia virus infection, since significant amounts of endocytic fluid-phase tracers were found in the lumen of the TGN, Golgi complex, and the wrapping cisternae. Using immunoelectron microscopy, we localized the vaccinia virus membrane proteins VV-p37, VV-p42, VV-p21, and VV-hemagglutinin (VV-HA) in large amounts in the wrapping cisternae, in the outer membranes of the IEV, and in the outermost membrane of the EEV. The bulk of the cellular VV-p37, VV-p21, and VV-p42 were in the TGN, whereas VV-HA was also found in large amounts on the plasma membrane and in endosomes. Collectively, these data argue that the TGN becomes enriched in vaccinia virus membrane proteins that facilitate the wrapping event responsible for the formation of the IEV.     

Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability

The fusion-associated small transmembrane (FAST) proteins of the fusogenic reoviruses are the only known examples of membrane fusion proteins encoded by non-enveloped viruses. While the involvement of the FAST proteins in mediating extensive syncytium formation in virus-infected and -transfected cells is well established, the nature of the fusion reaction and the role of cell-cell fusion in the virus replication cycle remain unclear. To address these issues, we analyzed the syncytial phenotype induced by four different FAST proteins: the avian and Nelson Bay reovirus p10, reptilian reovirus p14, and baboon reovirus p15 FAST proteins. Results indicate that FAST protein-mediated cell-cell fusion is a relatively non-leaky process, as demonstrated by the absence of significant [3H]uridine release from cells undergoing fusion and by the resistance of these cells to treatment with hygromycin B, a membrane-impermeable translation inhibitor. However, diminished membrane integrity occurred subsequent to extensive syncytium formation and was associated with DNA fragmentation and chromatin condensation, indicating that extensive cell-cell fusion activates apoptotic signaling cascades. Inhibiting effector caspase activation or ablating the extent of syncytium formation, either by partial deletion of the avian reovirus p10 ecto-domain or by antibody inhibition of p14-mediated cell-cell fusion, all resulted in reduced membrane permeability changes. These observations suggest that the FAST proteins do not possess intrinsic membrane-lytic activity. Rather, extensive FAST protein-induced syncytium formation triggers an apoptotic response that contributes to altered membrane integrity. We propose that the FAST proteins have evolved to serve a dual role in the replication cycle of these fusogenic non-enveloped viruses, with non-leaky cell-cell fusion initially promoting localized cell-cell transmission of the infection followed by enhanced progeny virus release from apoptotic syncytia and systemic dissemination of the infection.     

More than one door - Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes.

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

Mechanism of fusion of Sendai virus: role of hydrophobic interactions and mobility constraints of viral membrane proteins. Effects of polyethylene glycol

The mechanism of Sendai virus fusion was investigated by studying the effect of the dehydrating agent polyethylene glycol (PEG) on the interaction of the virus with erythrocyte membranes. The initial rate of virus fusion, monitored continuously by a fluorescence membrane fusion assay, increases approximately 5-fold in the presence of small amounts (4%, w/v) of PEG. The polymer did not trigger a massive nonspecific fusion event, as the limited number of virus particles that fuse per erythrocyte ghost remains unaltered. A mass action kinetic analysis reveals that the binding rate constant increases approximately 1.5-fold; however, the fusion rate constant is enhanced by about an order of magnitude. The results demonstrate that hydrophobic interaction forces dominate the actual fusion step of the virus. Below about 22 degrees C, the viral membrane proteins appear to be clustered, as revealed by temperature-dependent fluorescence measurements of fluorescently tagged viral proteins. Clustering is not modulated by the presence of PEG, and fusion at those conditions is not observed. It is concluded that in addition to hydrophobic interactions, constraints in the mobility of the viral membrane proteins codetermine the fusogenic capacity of the virus. Such constraints have to be relieved in order to allow the occurrence of the hydrophobic interactions. PEG primarily affects the surface properties of the viral membrane, including the properties of the membrane glycoproteins. We hypothesize that during virus-target membrane interaction but prior to the actual fusion reaction, the fusion protein may undergo a conformational change, triggered by an enhancement in hydrophobic environment, which accounts for the need to establish close, i.e. fusion-susceptible intermembrane contact between virus and target membrane.     

The amphiphilic membrane glycoproteins of Semliki forest virus are attached to the lipid bilayer by their COOH-terminal ends

The membrane location of the Semliki Forest virus glycoproteins E1, E2 and E3 was studied by protease treatment of (1) virus particles and (2) rough micro somes from cells infected with SF virus2. Protease treatment of virus particles removes all but the membrane-associated segments of the glycoproteins. Analyses of protease-treated SF virus membranes in 15% to 22.5% gradient acrylamide gels demonstrate the presence of three distinct peptide species with apparent molecular weights of 9000, 6000 and 5500. The 9000 and the 5500 molecular weight peptides have been aligned to the COOH-terminal end of E2 and the 6000 molecular weight peptide to the COOH-terminal end of El. The mapping of the peptides was done in a “Dintzis”-type of experiment (Dintzis, 1961) where we labelled the proteins of the virus with a gradient of [³⁵S]methionine increasing towards their COOH-terminal end.Protease treatment of microsomes from cells infected with SF virus removes only those parts of the viral glycoproteins that are transversing the lipid bilayer. Analyses of such treated membranes in sodium dodecyl sulphate-containing gels show that a 3000 molecular weight piece is digested from the COOH-terminal end of p62, the cellular precursor of E2 and E3. The COOH-terminus of p62 is shown to be equivalent to that of E2. These results thus demonstrate that the two amphiphilic membrane proteins of SF virus, E1 and E2 (p62) are attached to the lipid bilayer by their COOH-terminal ends. The COOH-terminal end of p62 (E2) spans the microsomal membrane. The third membrane protein, E3, probably does not interact with membrane lipids but is bound to the virus on E1 and (or) E2.