Involvement of the mutated M protein in altered budding polarity of a pantropic mutant, F1-R, of Sendai virus.

Wild-type Sendai virus buds at the apical plasma membrane domain of polarized epithelial MDCK cells, whereas a pantropic mutant, F1-R, buds at both the apical and basolateral domains. In F1-R-infected cells, polarized protein transport and the microtubule network are impaired. It has been suggested that the mutated F and/or M proteins in F1-R are responsible for these changes (M. Tashiro, J. T. Seto, H.-D. Klenk, and R. Rott, J. Virol. 67:5902-5910, 1993). To clarify which gene or mutation(s) was responsible for the microtubule disruption which leads to altered budding of F1-R, MDCK cell lines containing the M gene of either the wild type or F1-R were established. When wild-type M protein was expressed at a level corresponding to that synthesized in virus-infected cells, cellular polarity and the integrity of the microtubules were affected to some extent. On the other hand, expression of the mutated F1-R M protein resulted in the formation of giant cells about 40 times larger than normal MDCK cells. Under these conditions, the effects on the microtubule network were enhanced. The microtubules were disrupted and polarized protein transport was impaired as indicated by the nonpolarized secretion of gp80, a host cell glycoprotein normally secreted from the apical domain, and bipolar budding of wild-type and F1-R Sendai viruses. The mutated F glycoprotein of F1-R was transported bipolarly in cells expressing the F1-R M protein, whereas it was transported predominantly to the apical domain when expressed alone or in cells coexpressing the wild-type M protein. These findings indicate that the M protein of F1-R is involved in the disruption of the microtubular network, leading to impairment of cellular polarity, bipolar transport of the F glycoprotein, and bipolar budding of the virus.     

Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells.

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.     

Vesicular stomatitis virus glycoprotein does not determine the site of virus release in polarized epithelial cells

In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.     

Altered interaction between Sendai virus and a Chinese hamster cell mutant with defective cholesterol synthesis

An amphotericin B-resistant mutant (AMBr-1) isolated from the Chinese hamster V79 cell line is defective in a pathway for sterol synthesis and contains a much reduced free cholesterol level as compared with the parental V79. The character of the plasma membrane of AMBr-1 was compared with that of V79 by measuring the fusion with the envelope of the Sendai virus and also by measuring membrane fluidity: AMBr-1 was found to be more sensitive to Sendai virus-induced cytolysis than V79. Both assays for membrane-permeability change and electron spin resonance (ESR) study showed an enhanced response to the fusion between viral envelope and plasma membrane in AMBr-1 cells. Measurement of the fluorescence polarization for 1,6-diphenyl-1,3,5-hexatriene suggested that the membrane of AMBr-1 was more fluid than that of V79. This aberrant nature of the cell membrane of AMBr-1 might be caused by the altered membranous sterol content.     

Expression of herpes simplex virus glycoproteins in polarized epithelial cells.

Members of the herpesvirus family mature at inner nuclear membranes, although a fraction of the viral glycoproteins is expressed on the cell surface. In this study, we investigated the localization of herpes simplex virus type 2 (HSV-2) glycoproteins in virus-infected epithelial cells by using a panel of monoclonal antibodies directed against each of the major viral glycoproteins. All of the HSV-2 glycoproteins were localized exclusively on the basolateral membranes of Vero C1008, Madin-Darby bovine kidney, and mouse mammary epithelial cells. Using a monoclonal antibody to HSV-2 gD which cross-reacts with HSV-1 strains, we could also localize HSV-1 gD on the basolateral membranes of Madin-Darby bovine kidney cells. These results indicate that these molecules contain putative sorting signals that direct them to basolateral membrane domains.     

AIP1/Alix is a binding partner of Sendai virus C protein and facilitates virus budding

The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.     

The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells.

Human immunodeficiency virus type 1 (HIV-1) has been shown to exhibit a specific basolateral release in polarized epithelial cells. Previous investigators have used vaccinia virus recombinants expressing HIV proteins to demonstrate that virus release is nonpolarized in the absence of viral envelope glycoproteins. In this study, we developed a transient expression system which allows the use of Madin-Darby canine kidney polarized epithelial cells directly grown on semipermeable membranes. This procedure allowed us to investigate polarized HIV viral budding following introduction of proviral DNA constructs. Expression of env gene products in trans demonstrated the ability to polarize env-negative viruses in a dose-dependent manner. The targeting signal for polarized virus release was shown to be present in the envelope gp41 transmembrane protein and absent from the gp120 portion of env. At least part of this signal is within the gp41 intracytoplasmic domain. Mutants of the p17gag matrix protein were shown to be nonpolarized only when unable to interact with the envelope glycoproteins. Together, these data are consistent with a model of polarized virus budding in which capsid proteins, lacking a targeting signal, are targeted for specific basolateral release via an interaction of p17 with the envelope glycoprotein containing the polarization signal in its intracytoplasmic domain.     

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.     

Polycystin-1 is a microtubule-driven desmosome-associated component in polarized epithelial cells

In this study, we have analyzed the expression and localization of polycystin-1 in intestinal epithelial cells, a system lacking primary cilia. Polycystin-1 was found to be expressed in the epithelium of the small intestine during development and levels remained elevated in the adult. Dual-labelling indirect immunofluorescence revealed polycystin-1 at sites of cell-cell contact co-localizing with the desmosomes both in situ as well as in polarized Caco-2/15 cells. In unpolarized cultures of Caco-2/15 cells, polycystin-1 was recruited to the cell surface early during initiation of cell junction assembly. In isolated Caco-2/15 cells and HIEC-6 cell cultures, where junctional complexes are absent, polycystin-1 was found predominantly associated with the cytoskeletal elements of the intermediate filaments and microtubule networks. More precisely, polycystin-1 was seen as brightly labelled puncta decorating the keratin-18 positive filaments as well as the β-tubulin positive microtubules, which was particularly obvious in the lamellipodia. Treatment with the microtubule-disrupting agent, nocodazole, eliminated the microtubule association of polycystin-1 but did not seem to affect its association with keratin or the desmosomes. Taken together these data suggest that polycystin-1 is involved with the establishment of cell-cell junctions in absorptive intestinal epithelial cells and exploits the microtubule-based machinery in order to be transported to the plasma membrane.     

Gbetagamma recruits Rho1 to the site of polarized growth during mating in budding yeast

In mating mixtures of Saccharomyces cerevisiae, cells polarize their growth toward their conjugation partners along a pheromone gradient. This chemotropic phenomenon is mediated by structural proteins such as Far1 and Bem1 and by signaling proteins such as Cdc24, Cdc42, and Gbetagamma. The Gbetagamma subunit is thought to provide a positional cue that recruits the polarity establishment proteins, and thereby induces polarization of the actin cytoskeleton. We identified RHO1 in a screen for allele-specific high-copy suppressors of Gbetagamma overexpression, suggesting that Rho1 binds Gbetagamma in vivo. Inactivation of Rho1 GTPase activity augmented the rescue phenotype, suggesting that it is the activated form of Rho1 that binds Gbetagamma. We also found, in a pull-down assay, that Rho1 associates with GST-Ste4 and that Rho1 is localized to the neck and tip of mating projections. Moreover, a mutation in STE4 that disrupts Gbetagamma-Rho1 interaction reduces the projection tip localization of Rho1 and compromises the integrity of pheromone-treated cells deficient in Rho1 activity. In addition to its roles as a positive regulator of 1,3-beta-glucan synthase and of the cell integrity MAP kinase cascade, it was recently shown that Rho1 is necessary for the formation of mating projections. Together, these results suggest that Gbetagamma recruits Rho1 to the site of polarized growth during mating.     

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.     

Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells.

Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors.     

Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells

Epstein-Barr virus (EBV) initially enters the body through the oropharyngeal mucosa and subsequently infects B lymphocytes through their CD21 (CR2) complement receptor. Mechanisms of EBV entry into and release from epithelial cells are poorly understood. To study EBV infection in mucosal oropharyngeal epithelial cells, we established human polarized tongue and pharyngeal epithelial cells in culture. We show that EBV enters these cells through three CD21-independent pathways: (i) by direct cell-to-cell contact of apical cell membranes with EBV-infected lymphocytes; (ii) by entry of cell-free virions through basolateral membranes, mediated in part through an interaction between beta1 or alpha5beta1 integrins and the EBV BMRF-2 protein; and (iii) after initial infection, by virus spread directly across lateral membranes to adjacent epithelial cells. Release of progeny virions from polarized cells occurs from both their apical and basolateral membranes. These data indicate that multiple approaches to prevention of epithelial infection with EBV will be necessary.     

Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells.

Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.     

Protection of mice from wild-type Sendai virus infection by a trypsin-resistant mutant, TR-2.

A trypsin-resistant mutant of Sendai virus, TR-2, which could be activated by chymotrypsin but not by trypsin or the protease present in mouse lung, was inoculated intranasally into mice after being activated in vitro. TR-2 hardly brought about clinical illness or lung lesions in mice; the protease present in the lung could not activate the progeny virus, and the infection terminated after one-step replication. Nevertheless, the immunoglobulin A antibody against wild-type Sendai virus was produced in the respiratory tracts as well as the serum immunoglobulin G antibody, and the mice were protected from the challenge of the wild-type Sendai virus. On the basis of these results, TR-2 may provide a new model of live vaccine for paramyxoviruses; its availability as a live vaccine is also discussed.     

Sendai virus F glycoprotein induces IL-6 production in dendritic cells in a fusion-independent manner

We previously reported that inactivated Sendai virus particle (hemagglutinating virus of Japan envelope; HVJ-E) has anti-tumor effects by eliciting IL-6 production in dendritic cells (DCs). In the present study, we investigated which components of HVJ-E elicit IL-6 production. HVJ-E containing F0 protein inactive for virus envelope-cell membrane fusion enhanced IL-6 production. Reconstituted liposomes containing F protein stimulated IL-6 production. The antibody against F protein inhibited IL-6 secretion by HVJ-E. When carbohydrate chains of the F glycoprotein were removed, HVJ-E lost the ability to stimulate IL-6 secretion. These results suggest that F glycoprotein is required for IL-6 production in DCs.     

Lectin-resistant mutants of polarized epithelial cells.

Two lectin-resistant mutants derived from Madin Darby canine kidney cells, with constitutive alterations in the asparagine-linked carbohydrate moieties, retained the characteristic structural and functional epithelial polarity of the parental cells. A ricin-resistant cell line was unable to incorporate galactose-sialic acid into glycoproteins and, from the pattern of cross-resistance to other lectins, appears to be different from previously described lines resistant to this lectin: the mutation in a concanavalin A-resistant line results, probably, in the production of defective carbohydrate cores of glycoproteins. In spite of glycosylation defects which result in an increased electrophoretic mobility of many cellular glycoproteins, both mutants retained the typical asymmetric structure of the plasma membrane (microvilli on the apical surface, junctional elements on the basolateral surface), functional tight junctions, and unidirectional active transport of electrolytes and water. These results suggest that glycoproteins with terminal galactose-sialic acid moieties are not critically involved in the development and maintenance of polarity in epithelial cells. The mutant cells, particularly the ricin-resistant line, exhibited, however, morphological and electrophysiological changes which suggest a quantitative effect of the mutations on intracellular traffic of membranes and tight junction formation. The cell lines described in this paper, the first lectin-resistant mutants of epithelial lineage, should prove useful tools for studying the peculiarities of glycosylating pathways in polarized cells.     

Restricted viral RNA synthesis in establishment of persistent infection in Vero cells with a Sendai virus mutant

It was previously shown that a temperature-sensitive mutant of Sendai virus, ts-23, readily establishes persistent infection in Vero cells at 37 C, a permissive temperature for growth of the mutant. In the present study, it was demonstrated that the virus yield from ts-23-infected Vero cells at 37 C began to decrease 48 to 72 hr postinfection, after an initial phase of high virus production. Before the decrease in virus production, the formation of viral nucleoprotein declined, although synthesis of all species of viral protein continued. It was suggested that the limited formation of viral nucleoprotein and the decrease in virus production were due to the restriction of viral RNA synthesis which began to occur early after infection in ts-23-infected cells at 37 C. The mutant has a temperature-sensitive defect in RNA polymerase activity and the temperature 37 C, used for establishment of persistent infection, would be a semi-permissive temperature for the RNA polymerase activity of the mutant. The ts-23 mutant interfered with the replication of the parental wild virus in Vero cells at 37 C.     

Pneumopathogenicity of a Sendai virus protease-activation mutant, TCs, which is sensitive to trypsin and chymotrypsin.

A protease-activation mutant of Sendai virus, TCs, was isolated from a trypsin-resistant mutant, TR-5. TCs was activated in vitro by both trypsin and chymotrypsin. TCs was, however, less sensitive to trypsin and chymotrypsin than were the wild-type virus and TR-5, respectively. F protein of TCs had a single amino acid substitution at residue 114 from glutamine to arginine, resulting in the appearance of the new cleavage site for trypsin and the shift of the cleavage site for chymotrypsin. Activation of TCs in the lungs of mice occurred less efficiently than that of the wild type, and TCs caused a less severe pneumopathogenicity than did the wild-type virus, which supports our previous view that the in vitro trypsin sensitivity of Sendai virus can be a good indication of pneumopathogenicity in mice.