Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus.

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with budding virions were detected. Ferritin labeling and pulse-chase studies suggested a cell surface origin for these vacuoles. These experiments indicate that monensin inhibits the transport of Friend murine leukemia virus glycoproteins at an early stage, with a resultant block in the assembly and release of infectious virus.     

An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments.

The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.     

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.     

Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.     

Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darby canine kidney cells

Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus [VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins of the two viruses are synthesized simultaneously and assembled into virions at their characteristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane of the cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi cisternae. This indicates that the pathways of the two proteins towards the plasma membrane do not diverge before passage through the Golgi apparatus and therefore that critical sorting steps must take place during or after passage of the glycoproteins through this organelle. After its passage through the Golgi, the HA accumulated primarily at the apical membrane, where influenza virion assembly occurred. A small fraction of HA did, however, appear on the lateral surface and was incorporated into the envelope of budding VSV virions. Although predominantly found on the basolateral surface, significant amounts of G protein were observed on the apical plasma membrane well before disruption of the tight junctions was detectable. Nevertheless, assembly of VSV virions was restricted to the basolateral domain and in doubly infected cells the G protein was only infrequently incorporated into the envelope of budding influenza virions. These observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.     

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.     

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.     

Effect of monensin on the assembly of Uukuniemi virus in the Golgi complex.

The effect of the carboxylic ionophore monensin on the maturation of Uukuniemi virus, a bunyavirus, and the transport of its two membrane glycoproteins, G1 and G2, were studied in chicken embryo fibroblasts and baby hamster kidney cells. Virus maturation, which occurs in the Golgi complex (E. Kuismanen, K. Hedman, J. Saraste, and R. F. Pettersson, Mol. Cell. Biol. 2:1444-1458, 1982; E. Kuismanen, B. B?ng, M. Hurme, and R. F. Pettersson, J. Virol. 51:137-146, 1984), was effectively inhibited by the drug (1 or 10 microM) as studied by electron microscopy and by assaying the release of infectious or radiolabeled virus. Immunoelectron microscopy showed that association of viral nucleocapsids with the cytoplasmic surface of glycoprotein-containing Golgi membranes, a prerequisite for virus budding, was unaffected by monensin. In the presence of the drug, the virus glycoproteins assembled into long, tubular structures extending into the lumen of Golgi-derived vacuoles, suggesting that monensin inhibited a terminal step in the assembly of the virus. Intracellular transport and expression of the virus membrane glycoproteins G1 and G2 at the cell surface were not inhibited by monensin as studied by immunocytochemical and radiolabeling techniques. Pulse-chase experiments in the presence of monensin showed that intracellular G1 acquired only partially endo-H-resistant glycans. The sialylation of G1 appearing on the cell surface in the presence of the drug was decreased, whereas sialylation of G2 apparently was inhibited to a lesser extent, as shown by external labeling of the cells with the periodate-boro[3H]hydride method. Thus, monensin exerted a differential effect on the terminal glycosylation of G1 and G2. Unlike several membrane and secretory glycoproteins, both G1 and G2 could enter a functional transport pathway in the presence of monensin and become expressed at the cell surface.     

Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.     

Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8- nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.     

Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line

We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Analysis of the role of p200-containing vesicles in post-Golgi traffic.

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.     

Cell surface influenza haemagglutinin can mediate infection by other animal viruses.

We have used filter-grown Madin-Darby canine kidney (MDCK) cells to explore the mechanism by which influenza virus facilitates secondary virus infection. Vesicular stomatitis virus (VSV) and Semliki Forest virus (SFV) infect only through the basolateral surface of these polarized epithelial cells and not through the apical surface. Prior infection with influenza virus rendered the cell susceptible to infection by VSV or SFV through either surface. The presence of both a permissive and a restrictive surface for virus entry in the same cell allowed us to determine how the influenza infection enhanced the subsequent infection of a second virus. Biochemical and morphological evidence showed that influenza haemagglutinin on the apical surface serves as a receptor for the superinfecting virus by binding to its sialic acid-bearing envelope proteins. Influenza virus also facilitates secondary virus infection in non-epithelial cells; baby hamster kidney cells (BHK-21), which are normally resistant to infection by the coronavirus (mouse hepatitis virus MHV-A59), could be infected via the haemagglutinin-sialic acid interaction. Facilitation of secondary virus infection requires only the sialic acid-binding properties of the haemagglutinin since the uncleaved haemagglutinin could also mediate virus entry.     

Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.     

Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells

Enveloped viruses are excellent tools for the study of the biogenesis of epithelial polarity, because they bud asymmetrically from confluent monolayers of epithelial cells and because polarized budding is preceded by the accumulation of envelope proteins exclusively in the plasma membrane regions from which the viruses bud. In this work, three different experimental approaches showed that the carbohydrate moieties do not determine the final surface localization of either influenza (WSN strain) or vesicular stomatitis virus (VSV) envelope proteins in infected Madin-Darby Canine Kidney (MDCK) cells, as determined by immunofluorescence and immunoelectron microscopy, using ferritin as a marker. Infected concanavalin A- and ricin 1-resistant mutants of MDCK cells, with alterations in glycosylation, exhibited surface distributions of viral glycoproteins identical to those of the parental cell line, i.e., influenza envelope proteins were exclusively found in the apical surface, whereas VSV G protein was localized only in the basolateral region. MDCK cells treated with tunicamycin, which abolishes the glycosylation of viral glycoproteins, exhibited the same distribution of envelope proteins as control cells, after infection with VSF or influenza. A temperature-sensitive mutant of influenza WSN, ts3, which, when grown at the nonpermissive temperature of 39.5 degrees C, retains the sialic acid residues in the envelope glycoproteins, showed, at both 32 degrees C (permissive temperature) and 39.5 degrees C, budding polarity and viral glycoprotein distribution identical to those of the parental WSN strain, when grown in MDCK cells. These results demonstrate that carbohydrate moieties are not components of the addressing signals that determine the polarized distribution of viral envelope proteins, and possibly of the intrinsic cellular plasma membrane proteins, in the surface of epithelial cells.     

Effect of membrane protein on lipid bilayer structure: a spin-label electron spin resonance study of vesicular stomatitis virus.

Spin-label electron spin resonance (ESR) methods have been used to study the structure of the envelope of vesicular stomatitis virus (VSV). The data indicate that the lipid is organized in a bilayer structure. Proteolytic digestion of the glycoproteins which are the spike-like projections on the outer surface of the virus particle increases the fluidity of the lipid bilayer. Since the lipid composition of the virion reflects the composition of the host plasma membrane and the protein composition is determined by the viral genome, VSV was grown in both MDBK and BHK21-F cells to determine the effect of a change in lipid composition on the structure of the lipid bilayer of VSV. The lipid bilayer of the virion was found to be more rigid when derived from MDBK cells than from BHK21-F cells. Studies comparing spin-labeled intact cells and cell membrane fractions suggest that upon labeling the whole cell the spin label probes the plasma membrane. Comparison of spin-labeled VSV particles and their host cells indicates that the lipid bilayer of the plasma membrane is considerably more fluid than that of the virion. These results are discussed in terms of the effect of membrane-associated protein on the structure of the lipid bilayer.