Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Redistribution of cellular and herpes simplex virus proteins from the trans-golgi network to cell junctions without enveloped capsids

Herpes simplex virus (HSV) and other alphaherpesviruses assemble enveloped virions in the trans-Golgi network (TGN) or endosomes. Enveloped particles are formed when capsids bud into TGN/endosomes and virus particles are subsequently ferried to the plasma membrane in TGN-derived vesicles. Little is known about the last stages of virus egress from the TGN/endosomes to cell surfaces except that the HSV directs transport of nascent virions to specific cell surface domains, i.e., epithelial cell junctions. Previously, we showed that HSV glycoprotein gE/gI accumulates extensively in the TGN at early times after infection and also when expressed without other viral proteins. At late times of infection, gE/gI and a cellular membrane protein, TGN46, were redistributed from the TGN to epithelial cell junctions. We show here that gE/gI and a second glycoprotein, gB, TGN46, and another cellular protein, carboxypeptidase D, all moved to cell junctions after infection with an HSV mutant unable to produce cytoplasmic capsids. This redistribution did not involve L particles. In contrast to TGN membrane proteins, several cellular proteins that normally adhere to the cytoplasmic face of TGN, Golgi, and endosomal membranes remained primarily dispersed throughout the cytoplasm. Therefore, cellular and viral membrane TGN proteins move to cell junctions at late times of HSV infection when the production of enveloped particles is blocked. This is consistent with the hypothesis that there are late HSV proteins that reorganize or redistribute TGN/endosomal compartments to promote virus egress and cell-to-cell spread.     

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.     

Vaccinia virus entry, exit, and interaction with differentiated human airway epithelia

Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors.     

Bidirectional entry of poliovirus into polarized epithelial cells.

The interactions of viruses with polarized epithelial cells are of some significance to the pathogenesis of disease because these cell types comprise the primary barrier to many virus infections and also serve as the sites for virus release from the host. Poliovirus-epithelial cell interactions are of particular interest since this virus is an important enteric pathogen and the host cell receptor has been identified. In this study, poliovirus was observed to adsorb to both the apical and basolateral surfaces of polarized monkey kidney (Vero C1008) and human intestinal (Caco-2) epithelial cells but exhibited preferential binding to the basolateral surfaces of both cell types. Localization of the poliovirus receptor by a receptor-specific monoclonal antibody (D171) revealed a similar distribution predominantly on basolateral membranes, and treatment of cells with antibody D171 inhibited virus adsorption to both membrane surfaces. Poliovirus was able to initiate infection with similar efficiency following adsorption to either surface, and infection was blocked at both surfaces by D171, indicating that functional receptor molecules are expressed on both surfaces at sufficient density to mediate efficient infection at the apical and basolateral plasma membranes. Poliovirus infection resulted in a decrease in transepithelial resistance which was inhibited by prior treatment with monoclonal antibody D171 and occurred prior to other visible cytopathic effects. These results have interesting implications for viral pathogenesis in the human gut.     

Plasmalemmal vesicle associated protein (PV1) modulates SV40 virus infectivity in CV-1 cells

Plasmalemmal vesicle associated protein (Plvap/PV1) is a structural protein required for the formation of the stomatal diaphragms of caveolae. Caveolae are plasma membrane invaginations that were implicated in SV40 virus entry in primate cells. Here we show that de novo Plvap/PV1 expression in CV-1 green monkey epithelial cells significantly reduces the ability of SV40 virus to establish productive infection, when cells are incubated with low concentrations of the virus. However, in presence of high viral titers PV1 has no effect on SV40 virus infectivity. Mechanistically, PV1 expression does not reduce the cell surface expression of known SV40 receptors such as GM1 ganglioside and MHC class I proteins. Furthermore, PV1 does not reduce the binding of virus-like particles made by SV40 VP1 protein to the CV-1 cell surface and does not impact their internalization when cells are incubated with either high or low VLP concentrations. These results suggest that PV1 protein is able to block SV40 infectivity at low but not at high viral concentration either by interfering with the infective internalization pathway at the cell surface or at a post internalization step.     

Dimerization of the transmembrane domain of human tetherin in membrane mimetic environments

Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes.     

Role of receptor-mediated endocytosis in the formation of vaccinia virus extracellular enveloped particles

Infectious intracellular mature vaccinia virus particles are wrapped by cisternae, which may arise from trans-Golgi or early endosomal membranes, and are transported along microtubules to the plasma membrane where exocytosis occurs. We used EH21, a dominant-negative form of Eps15 that is an essential component of clathrin-coated pits, to investigate the extent and importance of endocytosis of viral envelope proteins from the cell surface. Several recombinant vaccinia viruses that inducibly or constitutively express an enhanced green fluorescent protein (GFP)-EH21 fusion protein were constructed. Expression of GFP-EH21 blocked uptake of transferrin, a marker for clathrin-mediated endocytosis, as well as association of adaptor protein-2 with clathrin-coated pits. When GFP-EH21 was expressed, there were increased amounts of viral envelope proteins, including A33, A36, B5, and F13, in the plasma membrane, and their internalization was inhibited. Wrapping of virions appeared to be qualitatively unaffected as judged by electron microscopy, a finding consistent with a primary trans-Golgi origin of the cisternae. However, GFP-EH21 expression caused a 50% reduction in released enveloped virions, decreased formation of satellite plaques, and delayed virus spread, indicating an important role for receptor-mediated endocytosis. Due to dynamic interconnection between endocytic and exocytic pathways, viral proteins recovered from the plasma membrane could be used by trans-Golgi or endosomal cisternae to form new viral envelopes. Adherence of enveloped virions to unrecycled viral proteins on the cell surface may also contribute to decreased virus release in the presence of GFP-EH21. In addition to a salvage function, the retrieval of viral proteins from the cell surface may reduce immune recognition.     

Role of the cytoplasmic domains of viral glycoproteins in antibody-induced cell surface mobility.

We have investigated the role of the cytoplasmic domains of the influenza virus hemagglutinin (HA) and the parainfluenza virus type 3 (PI3) fusion (F) glycoproteins as a determinant of their ability to undergo antibody-induced redistribution on plasma membranes. The viral envelope genes were truncated in their cytoplasmic domains by using oligonucleotide-directed mutagenesis and expressed by using recombinant vaccinia viruses. In HeLa cells, the truncated HA (HAt), like the full-length HA, did not cap in response to specific antibody. In CV-1 cells, HAt showed patchy surface immunofluorescence with few caps, whereas full-length HA exhibited capping in many cells in response to bivalent antibody. Quantitation of cap formation indicated a sevenfold decrease in the frequency of capping of HAt in comparison with full-length HA. Similarly, truncated F also exhibited a significant decrease in cap formation in comparison with full-length F. These results indicate that the ability of influenza virus HA and PI3 F to undergo redistribution in response to bivalent antibody has been altered by truncation of the viral glycoproteins and suggest that capping may involve interactions between the cytoplasmic domain of the viral glycoproteins and host cell components.     

HIV-1 Accessory Protein Vpu Internalizes Cell-surface BST-2/Tetherin through Transmembrane Interactions Leading to Lysosomes

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, β-transducin repeat-containing protein (βTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a βTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.     

Mechanisms of cell entry by influenza virus

A wide range of viruses, including many human and animal pathogens representing various taxonomic groups, contain genomes that are enclosed in lipid envelopes. These envelopes are generally acquired in the final stages of assembly, as viruses bud from regions of the membrane of the infected cell at which virally encoded membrane proteins have accumulated. The viruses procure their membranes during this process and mature particles 'pinch off' from the cellular membranes. Under most circumstances, initiation of another round of infection is dependent on two critical functions supplied by the envelope proteins. The virus must bind to cell-surface receptors of a new host cell, and fusion of the viral and cellular membranes must occur to transfer the viral genome into the cell. Enveloped viruses have evolved a variety of mechanisms to execute these two basic functions. Owing to their relative simplicity, studies of binding and fusion using enveloped viruses and their components have contributed significantly to the overall understanding of receptor-ligand interactions and membrane fusion processes - fundamental activities involved in a plethora of biological functions.     

Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane

Influenza virus acquires a lipid raft-containing envelope by budding from the apical surface of epithelial cells. Polarised budding involves specific sorting of the viral membrane proteins, but little is known about trafficking of the internal virion components. We show that during the later stages of virus infection, influenza nucleoprotein (NP) and polymerase (the protein components of genomic ribonucleoproteins) localised to apical but not lateral or basolateral membranes, even in cell types where haemagglutinin was found on all external membranes. Other cytosolic components of the virion either distributed throughout the cytoplasm (NEP/NS2) or did not localise solely to the apical plasma membrane in all cell types (M1). NP localised specifically to the apical surface even when expressed alone, indicating intrinsic targeting. A similar proportion of NP associated with membrane fractions in flotation assays from virus-infected and plasmid-transfected cells. Detergent-resistant flotation at 4 degrees C suggested that these membranes were lipid raft microdomains. Confirming this, cholesterol depletion rendered NP detergent-soluble and furthermore, resulted in its partial redistribution throughout the cell. We conclude that NP is independently targeted to the apical plasma membrane through a mechanism involving lipid rafts and propose that this helps determine the polarity of influenza virus budding.     

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.     

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.     

Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells.

Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. We have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, 125I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.     

Vaccinia virus preferentially enters polarized epithelial cells through the basolateral surface.

The uptake of vaccinia virus in polarized epithelial cells was studied to determine whether the site of entry was confined to either the apical or the basolateral membrane. Virus infection was monitored with a recombinant vaccinia virus carrying the luciferase reporter gene. Using cell lines MDCK and MDCK-D11, a clonal line with high transepithelial electrical resistance, we determined that vaccinia virus preferentially enters through the basolateral membrane. The possibility that there is a polarized cell surface distribution of vaccinia virus receptors which may be involved in systemic poxvirus infections is discussed.     

Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture

Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.     

Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.

In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes.     

Integrity of membrane lipid rafts is necessary for the ordered assembly and release of infectious Newcastle disease virus particles

Membrane lipid raft domains are thought to be sites of assembly for many enveloped viruses. The roles of both classical lipid rafts and lipid rafts associated with the membrane cytoskeleton in the assembly of Newcastle disease virus (NDV) were investigated. The lipid raft-associated proteins caveolin-1, flotillin-2, and actin were incorporated into virions, while the non-lipid raft-associated transferrin receptor was excluded. Kinetic analyses of the distribution of viral proteins in lipid rafts, as defined by detergent-resistant membranes (DRMs), in non-lipid raft membranes, and in virions showed an accumulation of HN, F, and NP viral proteins in lipid rafts early after synthesis. Subsequently, these proteins exited the DRMs and were recovered quantitatively in purified virions, while levels of these proteins in detergent-soluble cell fractions remained relatively constant. Cholesterol depletion of infected cells drastically altered the association of viral proteins with DRMs and resulted in an enhanced release of virus particles with reduced infectivity. Decreased infectivity was not due to effects on subsequent virus entry, since the extraction of cholesterol from intact virus did not significantly reduce infectivity. Particles released from cholesterol-depleted cells had very heterogeneous densities and altered ratios of NP and glycoproteins, demonstrating structural abnormalities which potentially contributed to their lowered infectivity. Taken together, these results indicate that lipid rafts, including cytoskeleton-associated lipid rafts, are sites of NDV assembly and that these domains are important for ordered assembly and release of infectious Newcastle disease virus particles.     

Cholesterol dependence of varicella-zoster virion entry into target cells

The entry of inhaled virions into airway cells is presumably the initiating step of varicella-zoster infection. In order to characterize viral entry, we studied the relative roles played by lipid rafts and clathrin-mediated transport. Virus and target cells were pretreated with agents designed to perturb selected aspects of endocytosis and membrane composition, and the effects of these perturbations on infectious focus formation were monitored. Infectivity was exquisitely sensitive to methyl-beta-cyclodextrin (M beta CD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited infection by enveloped, but not cell-associated, varicella-zoster virus (VZV) in a dose-dependent manner and exerted these effects on both target cell and viral membranes. Inhibition by M beta CD, which could be reversed by cholesterol replenishment, rapidly declined as a function of time after exposure of target cells to VZV, suggesting that an early step in viral infection requires cholesterol. No effect of cholesterol depletion, however, was seen on viral binding; moreover, there was no reduction in the surface expression or internalization of mannose 6-phosphate receptors, which are required for VZV entry. Viral entry was energy dependent and showed concentration-dependent inhibition by chlorpromazine, which, among other actions, blocks clathrin-mediated endocytosis. These data suggest that both membrane lipid composition and clathrin-mediated transport are critical for VZV entry. Lipid rafts are likely to contribute directly to viral envelope integrity and, in the host membrane, may influence endocytosis, evoke downstream signaling, and/or facilitate membrane fusion.