bafilomycin-a1 and alpha-sarcin

Feline calicivirus is a major causative agent of respiratory disease in cats. It is also one of the few cultivatable members of Caliciviridae. We have examined the entry process of feline calicivirus (FCV). An earlier study demonstrated that acidification in endosomes may be required. We have confirmed this observation and expanded upon it, demonstrating, using drugs to inhibit the various endocytic pathways and dominant-negative mutants, that FCV infects cells via clathrin-mediated endocytosis. We have also observed that FCV permeabilizes cell membranes early during infection to allow the co-entry of toxins such as alpha-sarcin. Inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked this permeabilization event, demonstrating that acidification is required for uncoating of the genome and access to the cytoplasm.

Topics: Acids; Animals; Antimalarials; Calicivirus, Feline; Cats; Cell Membrane; Chloroquine; Clathrin; Cytoplasm; Endocytosis; Endoribonucleases; Endosomes; Enzyme Inhibitors; Fungal Proteins; Genes, Dominant; Genome, Viral; Macrolides; Permeability; Protein Synthesis Inhibitors; Proton-Translocating ATPases; rab5 GTP-Binding Proteins; Receptors, Virus; RNA, Viral; Virus Replication

Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.

Topics: Adenoviruses, Canine; Animals; Anti-Bacterial Agents; Antibodies, Viral; Biological Transport; Cell Cycle; Cell Membrane; Cell Membrane Permeability; Cell Nucleus; Clathrin; Coated Pits, Cell-Membrane; Dextrans; Dogs; Dynamins; Endocytosis; Endoribonucleases; Enzyme Inhibitors; Fungal Proteins; GTP Phosphohydrolases; Intracellular Fluid; Macrolides; Microscopy, Electron; Mutagenesis; Neutralization Tests; Parvovirus, Canine; Proton-Translocating ATPases; Time Factors; Transferrin; Virion

Inhibitors of vacuolar proton-ATPase activity (5 microM bafilomycin A1 or 50 nM concanamycin A) prevented infection by reovirus particles but not by infectious subviral particles (ISVPs). Neither compound affected virus attachment or internalization. However, both compounds potently blocked cleavage of the viral protein mu 1C. Finally, both reovirus particles and ISVPs efficiently translocated the toxin alpha-sarcin to the cytosol during virus entry. Bafilomycin A1 blocked translocation of alpha-sarcin by reovirus particles but not by ISVPs.

Topics: Animals; Anti-Bacterial Agents; Capsid; Capsid Proteins; Cell Membrane Permeability; Endoribonucleases; Enzyme Inhibitors; Fungal Proteins; L Cells; Macrolides; Mice; Proton-Translocating ATPases; Reoviridae; Vacuoles