Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection.

BACKGROUND: Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3ß, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3ß during Dengue virus-2 (DENV-2) infection was investigated. METHODS: GSK3ß participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS: Phosphorylation of GSK3ß-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3ß reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3ß. CONCLUSIONS: The results suggest that GSK3ß participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection

BACKGROUND Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3β, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3β during Dengue virus-2 (DENV-2) infection was investigated. METHODS GSK3β participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS Phosphorylation of GSK3β-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3β reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3β. CONCLUSIONS The results suggest that GSK3β participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

Disentangling the role of PI3K/Akt, Rho GTPase and the actin cytoskeleton on dengue virus infection.

BACKGROUND: Infection generated by Dengue Virus (DENV) does not have a specific pharmacologic treatment. Therefore, it is necessary to investigate research strategies departing from traditional approaches. Studying cellular mechanisms during early DENV infection may allow the design of a host-based approach to antivirals. Herein, we describe early/late events of DENV infection in mammalian cells related to PI3K/Akt, Rho GTPases, and the actin cytoskeleton. METHODS: To evaluate whether PI3K/Akt/Rho GTPases and the actin cytoskeleton participate in DENV replication in Huh7 cells, chemical and genetic inhibition were performed over 24 h.p.i., including early (1-12 h.p.i.) and late (12-24 h.p.i.) infection. Effects were evidenced by quantification of viral titers, activation of kinases assayed by western blot and In-Cell Western and subcellular patterns registered by quantitative fluorescence microscopy. RESULTS: DENV infections induced activation of PI3K/Akt with concomitant reorganization of the actin cytoskeleton, which was confirmed using specific chemical inhibitors. Additionally, inhibition of PI3K/Akt/Rho GTPases and actin microfilaments significantly reduced new viral progeny. Blocking the downstream effectors (ROCK and Rac1) of this pathway mimicked the cellular phenotype of PI3K/Akt/Rho GTPases inhibition. Furthermore, blockage of the final executor (i.e., actin) of this cellular process in infected cells also elicited molecular and viral effects. Finally, combined PI3K/Akt inhibition and Rho GTPases knockdown (Rac1, Rac2 and Cdc42), showed a similar effect on DENV-2 titer to that observed by individual treatment. CONCLUSIONS: Taken together, these findings suggest that the PI3K/Akt pathway is involved in DENV-2 infection in a Rho GTPase- and actin-dependent manner and that DENV-2 uses this signaling cascade to efficiently replicate in cells.