Studies with two major cell types which are resistant to VSV infection reach opposite conclusions. It had been reported that macrophages promote Akt phosphorylation following experience of VSV but that Drosophila cells infected with VSV may actually downregulate Akt phosphorylation. We were interested in determining the interaction of VSV with the Akt PFT alpha signaling pathway to ascertain where the virus may communicate with the pathway. We found that in classically permissive cells, infection with VSV actively inhibits Akt activation in a manner determined by virus replication but that the accumulation of PIP3 is unhindered. It is particularly appropriate that VSV, currently being produced as an oncolytic virus, appears to have an original mechanism of blocking Akt signaling. Akt is really a transforming kinase, Posttranslational modification which will be often activated in cancer cells. MATERIALS AND Tissue culture and virus infections. BHK, HeLa, and Vero cells were cultured in Dulbeccos modified Eagles medium supplemented with 2 mM glutamine and seven days fetal bovine serum. HEK TERST and HEK TERV cell lines were cultured in MEM Alpha supplemented with 2 mM glutamine and 10 percent FBS. BSR T7/5 cells were cultured in Glasgow MEM supplemented with 1 mg/ml G418, 10% FBS, 2 mM glutamine, and 1 nonessential proteins. Cells were grown to 85 to 95-page confluence and then contaminated with VSV in growth medium at a multiplicity of infection of 10 PFU/cell. Membrane and cytosol fractionation. Cytosolic and membrane fractionation were primarily performed as described previously. Cells were collected on ice, and all procedures were performed at 4 C. Cells were gently washed once with ice cold phosphate buffered saline and then scraped in to homogenization buffer containing 25 mM Tris HCl, 2 mM EDTA, met inhibitors 10 mM NaCl, and 0. As directed by producer, 25 M sucrose and supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail. The cells were allowed to swell on ice for 10 min and then homogenized with 25 strokes of a glass homogenizer. Cell lysates were collected and centrifuged at 2,000 g for 5 min at 4 C, supernatants were then centrifuged at 100,000 g for 30 min, and the resulting supernatant was used as the cytosolic fraction. The pellet was carefully rinsed with PBS three times and removed with homogenization buffer containing hands down the Triton X 100 for 30 min. The Triton X 100 soluble portion was centrifuged at 14,000 g for 20 min at 4 C, and the resulting supernatant was used whilst the membrane fraction. Protein concentrations were determined by the Bio Rad protein assay using bovine serum albumin as a standard. Immunoblotting and recognition. Infected or mock infected cells were lysed in 35 mm 6 well dishes for 5 min at 4 C using 250 l of NP 40 lysis buffer supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail as directed by producer.