Ying Ray Lee

Autophagic machinery activated by dengue virus enhances virus replication

n Abstractn n Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.n n

Dengue virus infection induces autophagy: an in vivo study

BackgroundWe and others have reported that autophagy is induced by dengue viruses (DVs) in various cell lines, and that it plays a supportive role in DV replication. This study intended to clarify whether DV infection could induce autophagy in vivo. Furthermore, the effect of DV induced autophagy on viral replication and DV-related pathogenesis was investigated.Results and conclusionsThe physiopathological parameters were evaluated after DV2 was intracranially injected into 6-day-old ICR suckling mice. Autophagy-related markers were monitored by immunohistochemical/immunofluorescent staining and Western blotting. Double-membrane autophagic vesicles were investigated by transmission-electron-microscopy. DV non-structural-protein-1 (NS1) expression (indicating DV infection) was detected in the cerebrum, medulla and midbrain of the infected mice. In these infected tissues, increased LC3 puncta formation, LC3-II expression, double-membrane autophagosome-like vesicles (autophagosome), amphisome, and decreased p62 accumulation were observed, indicating that DV2 induces the autophagic progression in vivo. Amphisome formation was demonstrated by colocalization of DV2-NS1 protein or LC3 puncta and mannose-6-phosphate receptor (MPR, endosome marker) in DV2-infected brain tissues. We further manipulated DV-induced autophagy by the inducer rapamycin and the inhibitor 3-methyladenine (3MA), which accordingly promoted or suppressed the disease symptoms and virus load in the brain of the infected mice.We demonstrated that DV2 infection of the suckling mice induces autophagy, which plays a promoting role in DV replication and pathogenesis.

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

BackgroundWe previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.ResultsWe demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH4Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.ConclusionsIn this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

BackgroundMutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors.MethodsSuppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-rasval12 up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7–4) harboring the inducible Ha-rasval12 oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters.ResultsRetinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-rasval12 up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells “7-4” by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model.ConclusionsWe confirmed that RbAp46 is a Ha-rasval12 up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.

Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo

Dengue virus (DENV) utilizes the endoplasmic reticulum (ER) for replication and assembling. Accumulation of unfolded proteins in the ER lumen leads to ER stress and unfolded protein response (UPR). Three branches of UPRs temporally modulated DENV infection. Moreover, ER stress can also induce autophagy. DENV infection induces autophagy which plays a promotive role in viral replication has been reported. However, the role of ER stress in DENV-induced autophagy, viral titer, and pathogenesis remain unclear. Here, we reveal that ER stress and its downstream UPRs are indispensable for DENV-induced autophagy in various human cells. We demonstrate that PERK-eIF2α and IRE1α-JNK signaling pathways increased autophagy and viral load after DENV infection. However, ATF6-related pathway showed no effect on autophagy and viral replication. IRE1α-JNK downstream molecule Bcl-2 was phosphorylated by activated JNK and dissociated from Beclin 1, which playing a critical role in autophagy activation. These findings were confirmed as decreased viral titer, attenuated disease symptoms, and prolonged survival rate in the presence of JNK inhibitor in vivo. In summary, we are the first to reveal that DENV2-induced ER stress increases autophagy activity, DENV replication, and pathogenesis through two UPR signaling pathways both in vitro and in vivo.

Honeysuckle aqueous extract and induced let-7a suppress dengue virus type 2 replication and pathogenesis

ETHNOPHARMACOLOGICAL RELEVANCEnHoneysuckle (Lonicera japonica Thunb.), a traditional Chinese herb, has widely been used to treat pathogen infection. However, the underlying-mechanism remains elusive.nnnAIMS OF THE STUDYnTo reveal the host microRNA (miRNA) profile with the anti-viral activity after honeysuckle treatment.nnnMATERIALS AND METHODSnHere we reveal the differentially expressed miRNAs by Solexa® deep sequencing from the blood of human and mice after the aqueous extract treatment. Among these overexpressed innate miRNAs both in human and mice, let-7a is able to target the NS1 region (nt 3313-3330) of dengue virus (DENV) serotypes 1, 2 and 4 predicated by the target predication software.nnnRESULTSnWe confirmed that let-7a could target DENV2 at the predicated NS1 sequence and suppress DENV2 replication demonstrated by luciferase-reporter activity, RT-PCR, real-time PCR, Western blotting and plaque assay. ICR-suckling mice consumed honeysuckle aqueous extract either before or after intracranial injection with DENV2 showed decreased levels of NS1 RNA and protein expression accompanied with alleviated disease symptoms, decreased virus load, and prolonged survival time. Similar results were observed when DENV2-infected mice were intracranially injected with let-7a.nnnCONCLUSIONnWe reveal that honeysuckle attenuates DENV replication and related pathogenesis in vivo through induction of let-7a expression. This study opens a new direction for prevention and treatment of DENV infection through induction of the innate miRNA let-7a by honeysuckle.