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The prosurvival function of interferon-gamma-inducible GTPase (IRGM3) in coxsackievirus B3 infection

University of British Columbia

Interferon-γ-inducible GTPase (IGTP, or IRGM3) is a p47 GTPase upregulated in coxsackievirus B3 (CVB3)-infected murine heart and inhibits CVB3-induced apoptosis through activation of the PI3 kinase/Akt pathway. However, the mechanism of this PI3K/Akt activation and other prosurvival functions of IGTP are unknown. In this study, using a doxcycycline-inducible Tet-On HeLa cell line that overexpresses IGTP, I have demonstrated that focal adhesion kinase (FAK) is phosphorylated in response to IGTP expression and that transfection of a dominant negative FAK (FRNK) blocks Akt activation. Furthermore, induction of IGTP promoted the NF-κB activation as evidenced by its enhanced nuclear translocation and increased transcriptional activity. However, FRNK transfection and PI3K inhibitor both blocked the IGTP-induced translocation and NF-κB activation. Moreover, silencing NF-κB with siRNAs significantly inhibited the phosphorylation of FAK and Akt. Finally, blocking this survival pathway by FRNK transfection or NF-κB siRNA reduced CVB3 replication and enhanced cell death during CVB3 infection. Taken together, these results suggest that FAK is a mediator upstream of PI3K/Akt and that NF-κB functions as a downstream effector. As viral infections including CVB3 can cause endoplasmic reticulum (ER) stress and activate a group of coordinated signal pathways termed ER stress response, additional prosurvival mechanisms of IGTP related to ER stress response was explored. It was demonstrated that IGTP expression suppressed either chemical- or CVB3-induced upregulation of GRP78, the ER stress marker. IGTP expression strongly inhibited the activation of both the PERK and ATF6 pathways of ER stress responses. The suppression of ER stress responses by IGTP also led to attenuated induction of proapoptotic genes CHOP and GADD34. These data were further supported by experiments using IGTP knockout mouse embryonic fibroblast cells, in which the ER stress response induced by CVB3 infection was not relieved after interferon-γ treatment due to the absence of IGTP. Moreover, blocking PI3K/Akt pathway with either the PI3K inhibitor or dominant negative Akt construct significantly diminished the inhibitory effect of IGTP on ER stress response as well as its prosurvival effect. Therefore, IGTP expression relieves the ER stress response via a PI3K/Akt dependent mechanism, which contributes to cell survival and host defense during CVB3 infection.

Text

2010-07-20

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/26674

Pathology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/