E amin ation of the phosphorylation level of Akt in the HAstV1 infected cells incubated with LY294002, wortmannin, triciribine, or MK2206 for 24 h showed that all but triciribine http://www.selleckchem.com/products/Sunitinib-Malate-(Sutent).html treatment effectively blocked the phosphoryl ation of Akt. In addition to the Akt mediated cascade, Rac1 is also known to be targeted by PI3K activation. Blocking Rac1 with 50 uM NSC23766, an inhibitor of Rac1 specific GEF, did not interfere with the infection. We also tested for the involvement of other signaling cascades. H89 blocks the activity of protein kinase A by competing for the ATP binding site of PKAs catalytic subunit. Y27632 inhibits Rho associating pro tein kinase. Neither inhibitor had an inhibitory effect on viral cap sid protein e pression, indicating that neither the PKA nor the Rho mediated pathway is significant for HAstV1 gene e pression.

Inhibitors that block Akt or Rac1 activation did not prevent the progression of infectious process The increase in Akt activation at 0. 25 and 0. 5 h post infection suggests that PI3K activation occurs at an early stage of infection. We also note that there is an increase of Akt phosphorylation at 8 hpi. To further e amine if PI3K activation is needed in the initial phase of infec tion, inhibitors of PI3K, Akt, or Rac1 were added at 0, 2, or 8 hpi, and the proportion of cells positive for viral capsid e pression was e amined by immunofluores cence. The Rac1 inhibitor NSC23766 did not block viral gene e pression at any time point. The PI3K inhibitors LY294002 and wortmannin were effective in diminishing viral gene e pression only when added at 0 or 2 hpi, at the time range of effectiveness similar to that of the ERK inhibitor.

Neither PI3K inhibitor was effective at 8 hpi. Although triciribine treated cells appeared to e hibit a lower proportion of infected cells, the difference from the control sample was not signifi cant. MK 2206, the other Akt inhibitor, did not affect viral gene e pression, suggesting that block ade of Akt had little effect on HAstV1 infection. None theless, the results showing blockade of infection by PI3K inhibitors added at 0 and 2 hpi are consistent with the increased phosphorylation of Akt at 15 and 30 min post infection seen in the Western blot, which marks the increased PI3K kinase activity at those early time points, and suggest that PI3K activation is important at the initial stage of infection.

Effects of kinase inhibitors on viral RNA replication The immunofluorescence detection of viral capsid protein offered a qualitative indication of whether a given kinase inhibitor affected the initiation of the infection processes leading to viral gene e pression. In order to more quantita tively measure the effect of the drugs on viral propagation, the amount of Cilengitide viral RNA produced in the cells at 24 hpi in the presence or absence of the drugs was mea sured by quantitative real time RT PCR.