Alterations in the intracellular levels of monovalent ions have been documented in cells infected by viruses representing several groups and appear to be involved in the termination of cellular macromolecular synthesis and/or in the development of cytopathic effects. It is likely that different viruses employ alternative strategies to induce alterations in cellular monovalent ion levels. Uptake (""""""""influx"""""""") and release (""""""""efflux"""""""") of Rb86+, a K+ tracer, and Na22+ will be measured in Sindbis virus-, Semliki forest virus-(SFV) and vesicular stomatitis virus-(VSV) infected cells. The cells will be incubated in the presence of ouabain, which specifically inhibits the Na+/K+ ATPase, furosemide or bumetanide, which specifically inhibit the Na+/K+/2Cl cotransport system, or amiloride, which inhibits the Na+/H+ exchange system to determine which of these ion transport systems are inhibited by these different viruses. Levels of the Na+/K+ ATPase in isolated plasma membranes will be quantitated by kinetic studies of the binding of H3-ouabain or by acidic polyacrylamide gel electrophoresis (HPC-PAGE). The mechanisms by which Sindbis virus, SFV and VSV stimulate hexose transport, block pinocytosis, alter membrane potential and inhibit the transport of certain amino acids will also be investigated to provide an overview of the effects of these lytic viruses on plasma membrane function. In addition, nucleotides will be quantitated in virus-infected cells to establish a possible role for modulation of nucleotide levels in the inhibition of cellular protein synthesis and in the regulation of hexose uptake. Ultraviolet irradiation and conditional and nonconditional viral mutants will be employed to investigate which viral products are involved in inducing effects on plasma membrane-associated transport systems. The effects of Sindbis virus, SFV and VSV on plasma membrane-associated transported systems during infection of invertebrate (mosquito) cells will also be studied. Mosquito cell clones which develop and those which fail to develop a cytopathic effect will be compared to investigate the role of the plasma membrane in the establishment of viral persistence.
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