title: Novel dengue virus inhibitor 4-HPR activates ATF4 independent of protein kinase R-like Endoplasmic Reticulum Kinase and elevates levels of eIF2α phosphorylation in virus infected cells.
abstract: Infections by dengue virus (DENV) are increasing worldwide, with an urgent need for effective anti-DENV agents. We recently identified N-(4-hydroxyphenyl) retinamide (4-HPR), an anti-DENV agent effective against all 4 serotypes of DENV in cell culture, and in a lethal mouse model for DENV infection (Fraser et al., 2014b). Although identified as an inhibitor of DENV non-structural protein 5 (NS5) recognition by host nuclear import proteins, the precise impact and mode of action of 4-HPR in effecting DENV clearance remains to be defined. Significantly, concurrent with decreased viral RNA and infectious DENV in 4-HPR-treated cells, we previously observed specific up-regulation of transcripts representing the Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) arm of the unfolded protein response (UPR) pathway upon 4-HPR addition. Here we pursue these findings in detail, examining the role of specific PERK pathway components in DENV clearance. We demonstrate that 4-HPR-induced nuclear localization of Activating Transcription Factor 4 (ATF4), a pathway component downstream from PERK, occurs in a PERK-independent manner, implying activation instead occurs through Integrated Stress Response (ISR) kinases. Significantly, ATF4 does not appear to be required for the antiviral activity of 4-HPR, suggesting transcriptional events induced by ATF4 do not drive the 4-HPR-induced antiviral state. Instead, we demonstrate that 4-HPR induces phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), a target of ISR kinases which controls translation attenuation, and confirm the importance of phosphorylated-eIF2α in DENV infection using guanabenz, a specific inhibitor of eIF2α dephosphorylation. This study provides the first detailed insight into the cellular effects modulated by 4-HPR in DENV-infected cells, critical to progressing 4-HPR towards the clinic.
authors.affiliation: Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia.
authors.forename: J E
authors.initials: JE
authors.lastname: Fraser
authors.affiliation: Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia.
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.affiliation: Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore, Singapore.
authors.forename: K W K
authors.initials: KW
authors.lastname: Chan
authors.affiliation: Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore, Singapore.
authors.forename: S G
authors.initials: SG
authors.lastname: Vasudevan
authors.affiliation: Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia. Electronic address: david.jans@monash.edu.
authors.forename: D A
authors.initials: DA
authors.lastname: Jans
mesh_terms: D051701:Activating Transcription Factor 4 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D003716:Dengue Virus / Q000187:drug effects* / Q000502:physiology*; D017313:Fenretinide / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D010766:Phosphorylation; D014176:Protein Biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013312:Stress, Physiological; D056811:Unfolded Protein Response / Q000187:drug effects / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D051701:Activating Transcription Factor 4; D000998:Antiviral Agents; D017313:Fenretinide; D034741:RNA, Small Interfering; D019892:eIF-2 Kinase; C081452:Atf4 protein, mouse
keywords: Antiviral; Dengue virus; Guanabenz; Integrated stress response; Translation attenuation; Unfolded protein response