The carboxylic ionophores monensin and nigericin, at concentrations higher than 10 and 6 auM, respectively, prevent the penetration of the Semliki Forest virus (SFV) genome into the cytosol ofbaby hamster kidney (BHK-21) cells and thereby inhibit viral replication. In the absence of inhibitors, the entry of SFV is known to proceed by adsorptive endocytosis in coated vesicles, followed by acid-triggered membrane fusion in intracellular vacuoles or lysosomes. The results show that binding of the virus to the cell surface, adsorptive endocytosis, and intracellular transport of viruses to the lysosomes are only marginally affected by the ionophores. No direct virucidal effect is observed, nor is the membrane fusion activity of the virus at low pH directly affected. Sequential addition of monensin and ammonium chloride (a nonrelated lysosomotropic inhibitor of SFV entry) indicates that both inhibitors affect the same step in the entry pathway. On the basis of these data and the known effects of carboxylic ionophores and lysosomotropic weak bases on cellular pH gradients, we conclude that monensin inhibits penetration by increasing the pH in endocytic vacuoles and lysosomes above pH 6, which is the pH threshold for the viral membrane fusion activity.Monensin and nigericin are carboxylic ionophores that intercalate into membranes and abolish proton gradients by electroneutral transmembrane exchange of protons for monovalent cations (1). When added to cells, monensin causes a variety of effects depending on cell-type, concentration, and time of incubation. These include inhibition of protein secretion, the transport of plasma membrane glycoproteins to the cell surface (2-4), serum low density lipoprotein uptake and degradation (5), membrane protein recycling (5, 6), and fluid-phase endocytosis (6). It has also been shown that monensin blocks the entry of vesicular stomatitis virus and diphtheria toxin into tissue culture cells (7,8).In this study we have examined the effects of monensin and nigericin on the entry of Semliki Forest virus (SFV) into baby hamster kidney (BHK-21) cells. This simple enveloped virus is internalized by an endocytic pathway that involves coated pits, coated vesicles, endosomes, and secondary lysosomes (9,10). Penetration of the viral genome into the cytosol occurs when the virus reaches an organelle with a pH < 6. The mildly acidic pH induces a potent membrane fusion activity in the virus that results in a fusion reaction between the viral membrane and the vacuolar membrane (11, 12). The nucleocapsids are thereby transferred into the cytoplasmic compartment. Nonfused viruses and viral membrane proteins are subsequently degraded in the lysosomes (10).Our results indicate that the principal effect ofmonensin (and possibly nigericin, for which our data is less complete) during virus entry is to inhibit the penetration ofthe viral nucleocapsid from the intracellular vacuoles into the cytosol. (13,14). The 32P-SFV had 50% of the radioactivity in the RNA and 50% in the phospholipid. Monensi...

The organization of intermediate-sized filaments (IF) of the cytokeratin type was studied in cultures of PtK2 cells in which typical IF structures are maintained during mitosis, using a monoclonal antibody (KG 8.13). This antibody reacts, in immunoblotting experiments, with the larger of the two major cytokeratin polypeptides present in these cells but, using standard immunofluorescence microscopy procedures, does not react with the cytokeratin filaments abundant in interphase cells, in striking contrast to various antisera and other monoclonal cytokeratin antibodies. In the same cell cultures, however, the antibody does react with cytokeratin filaments of mitotic and early postmitotic cells. The specific reaction with cytokeratin filaments of mitotic cells only is due to the exposure of the specific immunologic determinant in mitosis and its masking in interphase cells. Treatment of interphase cells with both Triton X-100 as well as with methanol and acetone alters the cytokeratin filaments and allows them to react with this monoclonal antibody. A similar unmasking was noted after treatment with buffer containing 2 M urea or low concentrations of trypsin. We conclude that the organization of cytokeratin, albeit still arranged in typical IF, is altered during mitosis of PtK2 cells.Intermediate-sized filaments (IF) j of vertebrates are cytoplasmic structures that are notoriously stable, both mechanically and chemically, and resist extractions in buffers of a broad range of ionic strengths and pH values (for reviews, see references 1-4). Different types of IF have been distinguished by subunit polypeptide composition and by immunological criteria and their specific expression has been related to routes of cell differentiation (1-3, 5-7). Yet they all share some common principles of morphology and homologies of amino acid sequence (1)(2)(3)(4)(8)(9)(10). In spite of their remarkable stability, IF may undergo redistribution of filaments as well as rearrangements of subunit proteins in living cells. It has been described that arrays of vimentin filaments are reorganized during mitosis (11-15) and drug-induced perinuclear aggregation (11, 16-17) and similar observations have been made for cytokeratin IF in mitosis of some epithelial cells (6,12,18,19). In most cases such re-distributions have been inter- preted as altered distributions of intact IF that do not involve intrafilamentous changes such as disassembly and re-assembly of IF subunits, in agreement with electron microscopic observations of normaMooking IF in all stages of mitosis of various cultured cells of mesenchymal (11, 13, 15) and epithelial (19) origin. By contrast, certain epithelial cells exhibit a drastic, transient change of IF organization during mitosis in which IF are unravelled into different, yet still insoluble and polymeric structures that aggregate into variously sized, spheroidal masses containing cytokeratin (20)(21)(22). These observations suggest that the structural state of at least certain IF is physiologically regulated. In...

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