Alexandra Milena Cuartas-López1, Camilo Eduardo Hernández-Cuellar2, Juan Carlos Gallego-Gómez3. 1. Molecular and Translational Medicine Group, Medicine Faculty, Institute for Medical Research, Cra. 51 D No. 62 - 29, MUA building, Office 303, Universidad de Antioquia, Postal code: 050010, Medellín, Colombia. Electronic address: milecuartas@gmail.com. 2. Molecular and Translational Medicine Group, Medicine Faculty, Institute for Medical Research, Cra. 51 D No. 62 - 29, MUA building, Office 303, Universidad de Antioquia, Postal code: 050010, Medellín, Colombia. Electronic address: caehernandezcu@gmail.com. 3. Molecular and Translational Medicine Group, Medicine Faculty, Institute for Medical Research, Cra. 51 D No. 62 - 29, MUA building, Office 303, Universidad de Antioquia, Postal code: 050010, Medellín, Colombia. Electronic address: carlos.gallego@udea.edu.co.
Abstract
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.
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 DENVinfection may allow the design of a host-based approach to antivirals. Herein, we describe early/late events of DENVinfection 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.
Authors: Anh Phuong Luu; Zhenlan Yao; Sangeetha Ramachandran; Stephanie A Azzopardi; Linde A Miles; William M Schneider; H-Heinrich Hoffmann; Leonia Bozzacco; Gustavo Garcia; Danyang Gong; Robert Damoiseaux; Hengli Tang; Kouki Morizono; Charles M Rudin; Ren Sun; Vaithilingaraja Arumugaswami; John T Poirier; Margaret R MacDonald; Charles M Rice; Melody M H Li Journal: Viruses Date: 2021-10-20 Impact factor: 5.818