PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication

Xin

et al. 2019

J Virol

Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo. PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis. IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.

Section: Discussionmentioning

confidence: 99%

Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway

Xin

et al. 2019

J Virol

Genome Biology and Evolution

“…Interestingly, several previous studies have revealed that DENV causes the shutdown of host cell translation activities and in so doing protects cell survival ( Hou et al. 2017 ; Dionicio et al.…”

Section: Discussionmentioning

confidence: 99%

The transcriptional response of Aedes aegypti with variable extrinsic incubation periods for dengue virus

Koh

Allen

Herbert

et al. 2018

Dengue fever is the most prevalent arboviral disease globally. Dengue virus is transmitted primarily by the Aedes aegypti mosquito. One measure of the mosquito’s efficiency as a vector is the extrinsic incubation period (EIP), which is the time between the ingestion of viremic blood and the emergence of virions in the saliva. The longer it takes virus to infect the midgut and traverse to the saliva, the fewer opportunities the mosquito will have to transmit the pathogen over its lifetime. We have shown previously that EIP for dengue virus is highly heritable and that it is negatively correlated with vector lifespan. Here, we examined the transcriptional profiles for mosquitoes that varied in their EIP phenotype and identified pathways associated with either short or long EIP. We found that mosquitoes with short EIP have less active immune responses but higher levels of protein translation and calcium ion homeostasis and that mosquitoes with longer EIP may have slower metabolism. These findings indicate a complex interplay between calcium ion distribution, ribosome biogenesis, and metabolism and reveal potential pathways that could be modified to slow the rate of viral progression and hence limit lifetime transmission capability.

BioMed Research International

“…It was subsequently transferred onto an Immobilon-P Transfer Membrane (Millipore, Darmstadt, Germany). After blocking with 5% milk-TBS-0.1% Tween 20 buffer at RT for 1 h, the membrane was incubated with the indicated primary and secondary antibodies at RT for 1 h as the method done previously in this lab [ 29 ]. Specific primary antibodies included the 4G2 monoclonal antibody (a kind gift of Dr. Guey-Chuen Perng, National Cheng Kung University, Taiwan) for the dengue E protein and anti-actin mouse monoclonal antibody clone C4 (Merck Millipore, Burlington, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Specific strands of viral RNA were then amplified with the primer pair consisting of DV2-N1F (5′-CTGAAACGCGAGAGAA ACCG-3 ′ ) and DV2-N1R (5 ′ -GTATCCCTGCTGTTGG TGGG-3 ′ ). [29]. Specific primary antibodies included the 4G2 monoclonal antibody (a kind gift of Dr. Guey-Chuen Perng, National Cheng Kung University, Taiwan) for the dengue E protein and anti-actin mouse monoclonal antibody clone C4 (Merck Millipore, Burlington, MA, USA).…”

Section: Reverse Transcriptase-polymerase Chain Reaction (Rt-pcr)mentioning

confidence: 99%

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells

Cheng

Yang

et al. 2020

Self Cite

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.