Requirement of the vesicular system for membrane permeabilization by Sindbis virus

The vast majority of animal viruses enhance membrane permeability at two moments of infection. Herein, we describe that the entry of Sindbis virus (SV) in BHK cells promotes the co-entry of the macromolecule alpha-sarcin into the cytoplasm, thereby blocking translation. At a later stage, this protein toxin cannot enter the cell, while low molecular weight compounds, such as hygromycin B, readily pass through the plasma membrane of Sindbis virus-infected BHK cells. To unveil the participation of the different Sindbis virus structural proteins in late permeabilization, transfection experiments with each late gene by separate have been carried out. Our findings indicate that 6K is the main determinant that enhances membrane permeabilization. The co-expression of both viral glycoproteins employing a Sindbis virus variant that lacks the entire 6K gene partly modifies membrane permeability. Brefeldin A, a macrolide antibiotic that interferes with the proper functioning of the vesicular system, hampers the induction of membrane leakiness without significantly affecting viral protein synthesis. On the other hand, the flavone compound Ro-090179 also diminishes the entry of hygromycin B, while bafilomycin A1 or nocodazole have no effect. These data reveal the requirement of the vesicular system for late viral membrane permeabilization.     

Modification of membrane permeability induced by animal viruses early in infection

The infection of mouse L cells by encephalomyocarditis (EMC) virus renders the cell permeable to several translation inhibitors to which normal cells are impermeable. The modification of membrane permeability to the aminoglycoside antibiotic hygromycin B takes place early in infection during virus adsorption and also at the time when large amounts of viral coat proteins are synthesized. The specific inhibition of protein synthesis in virus-infected cells early in infection is also observed with other translation inhibitors such as anthelmycin, gougerotin, and edeine, indicating that a general permeabilization takes place during virus adsorption. The presence of 1 mM hygromycin B in the culture medium during the first hour of EMC infection strongly reduced the production of new infectious virus. In order to understand the molecular mechanisms involved in membrane permeabilization early during EMC infection, the cells were treated with compounds known to disrupt microtubules and microfilaments and to block pinocytotic processes, e.g., vinblastine, cytochalasin B, and colchicine. However, even in the presence of those compounds the early leakiness phenomenon was still observed. The presence of concanavalin A, which binds to certain cell surface recptors, or inhibitors that block energy production (e.g., FCCP and NaNs) had no significant effects on the modification of membrane permeability. Treatment of human cells with interferon does not prevent the increase in membrane permeability after EMC infection. No viral or cellular protein synthesis is necessary for the early membrane leakiness to occur, suggesting that virions themselves are responsible for this modification. The alteration of permeability to translation inhibitors is also observed after the infection of different mammalian cell lines with different viruses such as vesicular stomatitis virus and Semliki Forest virus, two lipid-enveloped RNA-containing viruses.     

Modification of membrane permeability in vaccinia virus-infected cells

Infection of mouse L cells with vaccinia virus leads to a modification in the permeability of the cell membrane. This modification permits the entry of the translation inhibitor hygromycin B (MW 550). On the basis that some inhibition of protein synthesis is also observed in the presence of the protein toxin α-sarcin (MW 16,800), it seems likely that macromolecules can also cross the membrane in vaccinia virus-infected cells. The modification of the cell membrane is observed very early during infection, in the first hour of viral adsorption. After this time the cell membrane gradually reseals, restoring its normal permeability characteristics. The multiplicities of infection needed to observe these modifications in permeability are rather low; the presence of 3 PFU/cell (150 physical particles/cell) are enough to promote the entry of the inhibitor hygromycin B. Ultraviolet (uv)-inactivated vaccinia virus (17,280 ergs/mm² promotes the increase in membrane permeability. Moreover, the presence of actinomycin D (25 μg/ml) and cordycepin (100 μg/ml) did not inhibit the passage of hygromycin B suggesting that a virion component is responsible for this phenomenon. That an intact virion is required to permeabilize L cells, is supported by the findings that neither purified core particles, nor virion surface proteins can induce the inhibition of translation by hygromycin B. Since interferon treatment appears to induce chemical, physical, morphological, and immunological alterations in the cell surface, the effect of interferon on changes in membrane permeability following vaccinia virus infection was investigated. We find that the permeability of the cell membrane is enhanced in interferon-treated, vaccinia virus-infected cells when compared to untreated, infected cells.     

Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts

In Sindbis virus-infected avian cells the development of the cytopathic effect is correlated with the disruption of plasma membrane function. Sindbis virus inhibits the activity of the Na+K+ATPase, a membrane-associated enzyme complex which regulates intracellular monovalent cation levels. Tunicamycin, which blocks envelope protein glycosylation, prevents inhibition of Na+K+ATPase activity and the development of morphological changes in Sindbis virus-infected cells. Although inhibition of Na+K+ATPase activity is not essential for the termination of host protein synthesis, membrane-mediated events may favor the selective translation of viral proteins. The termination of host protein synthesis does not contribute to the development of these cytopathic changes in the time frame examined. In tunicamycin-treated, Sindbis virus-infected cells, unglycosylated E1 is inserted into the plasma membrane but virus release is prevented. In productively infected cells, therefore, the inhibition of Na+K+ATPase activity and the development of the cytopathic effect may result from terminal events in virus assembly and/or virus release.     

Increased sensitivity of virus-infected cells to inhibitors of protein synthesis does not correlate with changes in plasma membrane permeability

Semliki Forest virus-infected BHK cells or herpes simplex virus-infected Vero cells were incubated with the protein synthesis inhibitors hygromycin B and gougerotin. Infected cells take up no more [3H]hygromycin or [3H]gougerotin than do mock-infected cells, at a time p.i. at which either compound is more inhibitory to protein synthesis in infected, than in mock-infected cells. The concentrations of hygromycin and gougerotin required to inhibit protein synthesis in intact cells (irrespective of whether they are infected or not) are several orders of magnitude higher than those required in either permeabilized cells or in cell-free systems. Infected cells take up 86Rb+ at the same rate as mock-infected cells, their intracellular content of K+ is the same, and the activity of the Na+ pump is the same. It is concluded that the increased efficacy of hygromycin and gougerotin in virus-infected cells is a consequence of altered intracellular compartmentation and that increases in permeability of the plasma membrane, if any, are so small as to be undetectable by direct methods.     

Myxoviruses do not induce non-specific alterations in membrane permeability early on in infection

Summary The permeability characteristics of cells infected with myxoviruses have been studied by measuring the concentrative uptake of nutrients, the concentration of intracellular K⁺, and the maintenance of the Na⁺ gradient across the plasma membrane. Cells either show no change at all (Sendai virus-infected BHK cells and measles virus-infected Vero cells) or they show a decreased ability to concentrate nutrients, while intracellular K⁺ and the Na⁺ gradient remain unchanged (Sendai and influenza virus-infected L-1210 cells, measles virus-infected lymphocytes and mumps virus-infected L-41 cells). In no case, therefore, was a change observed that resembles the non-specific increase in membrane permeability induced by haemolytic paramyxoviruses (35, 42) or the non-specific membrane leakiness postulated to take place in infected cells (8, 9). A preliminary account of some of these findings has been presented (39).With 5 Figures     

Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.     

Differential effects of ouabain on host- and sindbis virus-specified protein synthesis.

Ouabain, a specific inhibitor of the membrane-associated Na⁺/K⁺-ATPase (sodium pump), has a differential effect on protein synthesis in Sindbis virus-infected and uninfected chick embryo fibroblast (CEF) cultures. At low concentrations, cell-specified protein synthesis is inhibited while Sindbis virus-directed protein synthesis is stimulated. At higher ouabain concentrations, which inhibit protein synthesis in uninfected CEF cells 95% or more, viral protein synthesis continues. Ouabain treatment of uninfected CEF cells raises the intracellular Na⁺ concentration and lowers the intracellular K⁺ concentration. The effect of Sindbis virus infection is similar. The changes in intracellular Na⁺ and K⁺ concentrations appear responsible for the selective inhibition of the translation of host cell mRNAs in Sindbis virus-infected or ouabain-treated CEF.     

Antiviral Immune Cytolysis at an Early Stage of Paramyxovirus Infection

Antiviral antibody and rabbit complement added as early as 5 min after infection, and with relatively low virus/cell multiplicity, lysed mouse ascites lymphoma cells infected with Sendai or Newcastle disease virus. Inactive Sendai virus at much higher input also sensitized ascites cells and mouse fibroblast monolayers to early antiviral immune cytolysis. At 4 C where adsorption but no penetration occurred, antibody removed virus from the cell membrane and little cytolysis was observed. The ascites cells were sensitive to antibody and complement at all times after the start of penetration and uncoating, indicating that viral envelope antigen is constantly present on the cell membrane. Significant cross-reactions by immune cytolysis between Newcastle disease virus- and Sendai virus-infected cells suggested possible participation of host antigens of the viral envelope. No comparable antiviral immune cytolysis was observed with influenza strains PR8 and WSN. Cell viability was estimated by dye exclusion and the ability to form acid from glucose as indicated by colorimetric pH of the medium. The relation of antiviral immune cytolysis to changes in the membrane resulting in cell fusion is considered.     

Palmitoylation of CM2 is dispensable to influenza C virus replication

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.     

L929 cells infected with temperature sensitive mutants of vesicular stomatitis virus: virus replication is necessary for induction of changes in membrane permeability

Summary Infection of L₉₂₉ murine cells with vesicular stomatitis virus (VSV) results in inhibition of host protein synthesis and appearance of membrane alterations at a time when cells are still actively engaged in viral protein synthesis. VSV temperature-sensitive (ts) mutants have been used to explore the role(s) played by the virus-coded proteins in the genesis of these effects. Cells were infected with each of fivets mutants representing the known complementation groups of VSV Indiana serotype, and incubated at permissive (32 °C) and non-permissive temperatures (39 °C). Protein synthesis in the presence and absence of Hygromycin B (Hyg.B) was analyzed during virus infection via incorporation of³⁵S-methionine in acid-precipitable material and SDS-polyacrylamide gel electrophoresis.Data indicate that mutants belonging to groups I (L protein), II (NS protein) and IV (N protein) do not inhibit host protein synthesis and do not induce any membrane changes when grown at the non-permissive temperature. Mutants of group III (M protein) and V (G protein), instead, do inhibit cell protein synthesis and induce membrane changes also when grown at the non-permissive temperature; this suggests that these effects do not correlate with the biological activity of these proteins and their interaction with the cellular membrane. On the other hand, mutants exhibiting defective steps of nucleocapsid replication are apparently unable to induce these effects once more suggesting that virus replication per se is essential, as also indirectly shown by experiments employing cycloheximide to mimic shut-off.This work was supported in part by grant from Consiglio Nazionale delle Ricerche, Rome, Italy, No. 85.00842.04 (Gruppo di Virologia).     

Selective inhibition of cellular protein synthesis by amphotericin B in EMC virus-infected cells

The addition of amphotericin B to EMC-infected cells induces a selective inhibition of cellular as compared to viral protein synthesis. A concentration of 5 μM amphotericin B in the culture medium produced 80% inhibition of actin synthesis, whereas the synthesis of the viral protein γ was inhibited by only 20%. The selective inhibition of cellular mRNA translation induced by this polyene antibiotic depends on the extracellular concentration of NaCl: In the absence of NaCl in the culture medium, the inhibition of actin synthesis was 25% in the presence of 10 μM amphotericin B, whereas, in hypertonic media, the inhibition of cellular protein synthesis was complete. These results indicate that the modification of membrane permeability by amphotericin B has a selective effect on the inhibition of cellular protein synthesis, and that the concentration of NaCl in the culture medium strongly influences the relative synthesis of cellular and viral proteins in EMC-infected cells.     

Covalent attachment of psoralen to a single site on vesicular stomatitis virus genome RNA blocks expression of viral genes

Mumps virus was adapted to growth in Vero cells, which yielded virus of high infectivity titers. The structural polypeptides of purified virions grown in Vero cells were similar to those described previously for egg-grown mumps virus: L (200K), HN (79K), NP (72K), F₁ (61K), P (45K), M (40K), and F₂ (16K). We have analyzed the synthesis of viral polypeptides in Vero cells by pulse labeling with radioactive amino acid precursors. The nucleoprotein (NP) was the first to be detected intracellularly above the cellular protein background at 6 h.p.i. By 12 h.p.i., all viral polypeptides were observed except for the glycoproteins F₁ and F₂, which are derived from a precursor designated F₀ (74K). Two low-molecular-weight polypeptides not present in purified virions were also detected in infected cells. They are designated pI (28K) and pII (19K). Peptide mapping revealed that these two polypeptides share regions of their amino acid sequence and that they are also related to the structural protein P. Polypeptides pI and pII were found in several cell types (Vero, CEF, MDBK cells) infected with mumps virus. Infection of Vero cells with other paramyxoviruses (SV5 and Sendai virus) did not induce the synthesis of proteins comparable to pI and pII, whereas in mumps virus-infected cells no counterpart to the nonstructural C protein of Sendai virus was detected. Pulse-chase experiments suggest that pI and pII may not be derived from P by proteolytic cleavage.     

Microscopy of internal structures of Sendai virus associated with the cytoplasmic surface of host membranes

The cytoplasmic surface (PS) of the plasma membrane of cells infected with Sendai virus was studied by immunofluorescence microscopy and freeze-drying electron microscopy. After cells had been attached to glass coverslips, they were subjected to a jet stream of physiological buffer which sheared off the upper portion of each cell, leaving the attached membrane with the PS exposed. This uncapping maneuver permitted direct examination of internal virus-specific elements associated with the inner surface of the host cell. At a stage of infection at which viral budding occurs, strands of nucleoprotein (RNP) were observed to be attached to the PS of plasma membranes. The sites at which RNP was adherent to the membrane were modified by virus-specific particles arranged in orthogonal patterns. The presence of the same crystalline structures in the hydrophobic domain of freeze-fractured membranes indicated that they were inserted into the inner lipid leaflet. The spatial association of the surface glycoprotein spikes and the internal RNP with this crystalline structure suggests its special relationship if not identity with the internal viral matrix (M) protein. The possible significance of the localization and crystalline nature of this structural element with respect to viral morphogenesis, hemolytic and cell-fusing activities is discussed. In contrast to the foregoing changes observed in the infected cell, no detectable viral antigens were found on the PS of normal cells to which exogenous virions had been fused. Absence of internalized antigen from the PS under these circumstances could indicate that infectious viral components are processed by the potential host cell in a manner which differs from what is observed with human erythrocytes. In the latter instance internalized antigens after fusion of virus to the cell remain associated with the PS.     

A cell-free model of the encephalomyocarditis virus-induced inhibition of host cell protein synthesis.

When preincubated extracts from Krebs-II cells were supplemented with total poly(A)-containing RNA isolated from encephalomyocarditis virus-infected Krebs-II cells, two phenomena characteristic of EMC virus infection of these cells in vivo were observed: (i) preferential translation of viral rather than cellular, mRNA, and (ii) a general inhibition of protein synthesis, relative to the synthesis in samples where the poly(A)-containing RNA from uninfected cells served as the template. The first effect could not be explained by an irreversible functional inactivation of cellular templates and seemed to result from a direct interference of viral RNA with the translation of cellular mRNA. The second effect was due primarily to the presence of double-stranded RNA.     

The polypeptides of influenza virus. VII. Synthesis of the hemagglutinin

Post-translational cleavage of the influenza viral glycoprotein HA occurs to different extents in different systems, varying not only for a particular virus strain grown in different host cells, but also for two strains of virus grown in the same host cell. Preparations of virus in which the HA is not substantially cleaved contain hemagglutinating and infectious virions. Cleavage occurs at different sites in the HA molecule of different virus strains.The hemagglutinin glycoprotein HA is always found in association with cytoplasmic membranes and becomes rapidly incorporated into plasma membranes. Following a 10-min pulse-label, there is already about half as much HA in preparations of plasma membranes as eventually accumulates there during a 90-min chase. Membrane preparations which appear to be mainly composed of smooth endoplasmic reticulum are greatly enriched for HA while plasma membranes contain HA and the other major viral proteins. At 24 hr after infection, the amount of HA or its cleavage products in BHK21 cells infected with Bel or WSN represents a much smaller proportion of the total viral protein than the proportion of HA in purified virions. The same is true for the membrane protein, M, whereas NP is present in excess in the infected cell.Inhibition of protein synthesis by puromycin stops the incorporation of glucosamine into Bel-infected HeLa cells almost immediately, suggesting that glycosylation of HA occurs quickly. However, fucose continues to be incorporated for apporoximately 10–15 min after protein synthesis has been blocked by puromycin or after glycosiliation has been inhibited by glucosamine hydrochloride.     

Antibody-Mediated Fusion of FL Amnion Cells Infected with Parainfluenza Virus Type 2

Addition of viral or cellular antiserum to FL amnion cells infected with type 2 parainfluenza virus caused the formation of polykaryons not observed during viral replication in the absence of antibody. This antibody-mediated fusion, which did not occur with Sendai virus-infected cells, is probably effected through close apposition of membrane segments bearing newly synthesized viral envelope constituents and does not directly involve glycoprotein receptors.     

Antibody-Mediated Fusion of FL Amnion Cells Infected with Parainfluenza Virus Type 2

Addition of viral or cellular antiserum to FL amnion cells infected with type 2 parainfluenza virus caused the formation of polykaryons not observed during viral replication in the absence of antibody. This antibody-mediated fusion, which did not occur with Sendai virus-infected cells, is probably effected through close apposition of membrane segments bearing newly synthesized viral envelope constituents and does not directly involve glycoprotein receptors.     

Inability of translation of mRNA for HVJ (Sendai virus) M protein in a rat glioma cell line at nonpermissive temperatures

Summary The synthesis of M protein of HVJ (Sendai virus) in rat glioma (C6) cells, as has previously been reported, is selectively reduced at a nonpermissive temperature of 39 °C. In this phenomenon no difference was found in the viral RNA synthesis and the stability of the viral RNAs between the permissive and nonpermissive temperatures. Oligo dT-selected RNA from the infected cells at either temperature similarly directed M protein synthesis in cell-free translation reactions even if the reaction was performed at 39°C. These results suggest that the restriction of M protein synthesis in C6 cells at the nonpermissive temperature might be due to the inability of translation apparatus to interact with the mRNA coding for M protein.