Hence, the activation of p38 and Akt pathways upon infection appears to be either non essential for HAstV1 infection or redundant with other pathways that could relay the essential signals for the infectious processes. It is interesting to note that wortmannin treatment showed no blockade of RNA replication, but e hibited a block in viral release. Immunofluorescent detection scientific research of viral capsid protein revealed that treatment with wortmannin caused unusual punctate staining of the capsid protein, which suggests that the reagent failed to block viral entry, but was effective in delaying the process leading to capsid e pression showing aberrant distribution.

The time point e amined for viral RNA replication, 24 hpi, may have been the point when viral RNA replication had already reached a plateau, but the inhibitory effect of wortmannin on the release of RNA and virion may have been visible because of the delay of the infectious process. Treatment with triciribine enhanced viral RNA replica tion in HastV1 infected cells, which possibly caused the increased viral release that was inferred from the level of viral RNA and capsid protein in the culture supernatant. Surprisingly, we found that the Akt phosphor ylation was not effectively blocked at 24 hpi and viral capsid release was enhanced in a dose dependent manner. We also noted that triciribine treatment slightly enhanced cell viability. Overall, the treatment appeared to have a positive effect on viral propagation in our e periments, rather than an inhibitory effect. Similarly, treatment with NSC23766 or Y27632 increased the e tent of viral RNA replication.

Interestingly, a marked increase in the phos phorylated Akt level was observed in cells treated with each drug. Akt activation is known to involve a feedback loop activating Rac1, led by ROCK inhibition using Y27632. Because Rho family sig naling events are known to involve balanced regulation, inhibition of another member of the Rho family, Rac1, by NSC23766 could also have activated such a feedback loop. The activated Akt possibly caused an in crease in protein synthesis, which could enhance viral RNA replication. We noted that two Akt phosphorylation inhibitors affect HAstV1 infection differently. Triciribine apparently increased the amount of viral RNA and the release of viral RNA and capsid in the culture supernatant, whereas MK2206 did not.

This difference could be due to a difference in the drugs inhibitory mechanisms. Triciribine inhibits Akt phosphorylation by binding to the PH domain of Akt, thereby blocking its recruitment to the plasma membrane, whereas MK2206 binds to the catalytic domain of Akt and inhibits its phosphor ylation. Triciribine is also known to inhibit GSK-3 cellular DNA synthesis. Nonetheless, neither Akt inhibitor blocked viral infection. In summary, our study has revealed that two signaling pathways, mediated by ERK and PI3K, are important for HAstV1 infection.