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Inhibition of protein kinase C promotes dengue virus replication.

ABSTRACT: Dengue virus (DENV) is a member of the Flaviviridae family, transmitted to human via mosquito. DENV infection is common in tropical areas and occasionally causes life-threatening symptoms. DENV contains a relatively short positive-stranded RNA genome, which encodes ten viral proteins. Thus, the viral life cycle is necessarily rely on or regulated by host factors.In silico analyses in conjunction with in vitro kinase assay were used to study kinases that potentially phosphorylate DENV NS5. Potential kinase was inhibited or activated by a specific inhibitor (or siRNA), or an activator. Results of the inhibition and activation on viral entry/replication and host cell survival were examined.Our in silico analyses indicated that the non-structural protein 5 (NS5), especially the RNA-dependent RNA polymerase (RdRp) domain, contains conserved phosphorylation sites for protein kinase C (PKC). Phosphorylation of NS5 RdRp was further verified by PKC in vitro kinase assay. Inhibitions of PKC by a PKC-specific chemical inhibitor or siRNA suppressed NS5 phosphorylation in vivo, increased viral replication and reduced viability of the DENV-infected cells. In contrary, activation of PKC effectively suppressed intracellular viral number.These results indicated that PKC may act as a restricting mechanism that modulates the DENV replication and represses the viral outburst in the host cells.

SUBMITTER: Noppakunmongkolchai W

PROVIDER: S-EPMC4774189 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Inhibition of protein kinase C promotes dengue virus replication.

Noppakunmongkolchai Warobon W Poyomtip Teera T Jittawuttipoka Thichakorn T Luplertlop Natthanej N Sakuntabhai Anavaj A Chimnaronk Sarin S Jirawatnotai Siwanon S Tohtong Rutaiwan R

Virology journal 20160301

<h4>Background</h4>Dengue virus (DENV) is a member of the Flaviviridae family, transmitted to human via mosquito. DENV infection is common in tropical areas and occasionally causes life-threatening symptoms. DENV contains a relatively short positive-stranded RNA genome, which encodes ten viral proteins. Thus, the viral life cycle is necessarily rely on or regulated by host factors.<h4>Methods</h4>In silico analyses in conjunction with in vitro kinase assay were used to study kinases that potenti ...[more]

PMID: 26931565

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Project description:UnlabelledDengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds-dihydrodibenzothiepines (DHBTs), identified through high-throughput screening-with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.ImportanceThe dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.

Project description:Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, ?-subunit of eukaryotic initiation factor 2 (eIF2?), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.

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Inhibition of protein kinase C promotes dengue virus replication.

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Inhibition of protein kinase C promotes dengue virus replication.

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