The entry of fowl plague virus, an avian influenza A virus, into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically . At low multiplicity and 0°C, viruses bound to the cell surface but were not internalized . Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37°C, part of the virus became resistant to removal by neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium . The neuraminidase-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein.When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0°C and, after warming at 37°C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles . Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures .

Our previous studies have shown that Semliki Forest virus (SFV), a togavirus, binds to cells in the cold and, when the temperature is raised to 37°C, the virus particles are rapidly internalized into endosomes and secondary lysosomes by adsorptive endocytosis (1-3) . Under normal conditions of infection, fusion between the plasma membrane and the virus envelope does not occur (4, 5) . We observed, however, that fusion between SFV and the plasma membrane could be artificially triggered by mildly acidic medium (pH 5 .0-6 .0) (4); as a result, the nucleocapsid entered the cell directly through the plasma membrane, and the spike glycoproteins were left on the cell surface (4) . Fusion with liposomes containing phospholipids and cholesterol as target membranes can also be induced by a brief drop in pH (6) . On the basis of these and other data, we have proposed that, under normal conditions of infection, the membrane fusion activity of SFV is needed for the penetration of the nucleocapsids through the lysosomal membrane, and that the low lysosomal pH is critical in triggering the reaction (references 1, 2, 4, and 6; footnote 1) .During the course of our studies on virus-cell fusion, we observed the formation of large polykaryons when BHK-21 cells were exposed to SFV and low pH . In some cases, these ' Helenius, A ., M . Marsh, and J . White. Manuscript in preparation. 674JUDY WHITE, KARL MATLIN, and ARI HELENIUS European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany ABSTRACT Representatives of three families of enveloped viruses were shown to fuse tissue culture cells together. These were : Semliki Forest virus (SFV, a togavirus), vesicular stomatitis virus (a rhabdovirus), and two myxoviruses, fowl plaque virus and Japan influenza virus (Japan/ A/305/57) . Unlike paramyxoviruses, whose fusion activity is known to occur over a broad pH range, fusion by these viruses was restricted to mildly acidic pH . The pH thresholds for the four viruses were 6.0, 6.1, 5.5, and 5 .1, respectively . The precursor form of Japan influenza, which is not infectious and which contains the uncleaved hemagglutinin, had no fusion activity . This result suggested a role for the influenza hemagglutinin in the low-pH-dependent membrane fusion activity . Taken together, our results show that low-pH-induced fusion is a widespread property of enveloped animal viruses and that it may play a role in the infective process.The fusion reactions with all four viruses were fast, efficient, and easy to induce . With UVinactivated SFV, the fusion was shown to be nonlytic and the polykaryons were viable for at least 12 h. 30 ng of SFV/1 x 106 BHK-21 cells were required for 50% fusion, and 250 ng sufficed to fuse the entire culture into a single polykaryon .encompassed the entire culture . In this paper we have characterized this phenomenon and we have demonstrated that, in addition to SFV, representatives of both the myxovirus and rhabdovirus families induced extensive cell fusion at low pH .

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Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical ␤1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a ␤1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of ␤1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane. INTRODUCTIONPolarization of cells is a fundamental process in biology. Epithelial cells polarize into apical and basolateral poles. Much has been learned recently about the mechanisms of epithelial polarization. Three major polarization complexes, the Par3/Par6/atypical protein kinase C (aPKC), Crumbs/ PATJ/Stardust, and Scribble/Discs Large/Lethal Giant Larvae complexes are conserved from Caenorhabditis elegans and Drosophila to mammals and are essential for epithelial polarization (Roh and Margolis, 2003;Macara, 2004). Cell polarization also requires interactions of the cells with each other and the extracellular matrix (ECM), as well as polarized organization of the cytoskeleton and membrane traffic, although how these processes are connected to the three polarization complexes is largely obscure (Vega-Salas et al., 1987;Yeaman et al., 1999;O'Brien et al., 2002;Mostov et al., 2003;Nelson, 2003;Zegers et al., 2003).Formation of epithelial tissues requires that the orientation of polarity of individual epithelial cells be coordinated in space and time. This was underappreciated in studies using cells grown on filter supports, because the filter provides an overriding extrinsic cue to orient the cells with the basolateral surface facing the filter and the apical surface opposite. Building on earlier observations (Wang et al., 1990a), we found that generation of epithelial polarity can be uncoupled from the orientation of that polarity (O'Brien et al., 2001). Madin-Darby canine kidney (MDCK) epithelial cells grown in three-dimensional (3D) collagen gels form cysts, where the apical surface of a polarized monolayer of cells fa...

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