Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection

Chu, Li Wei; Huang, Yi Lung; Lee, Jin Hui; Huang, Long Ying; Chen, Wei Jun; Lin, Ya Hsuan; Chen, Jyun Yu; Xiang, Rui; Lee, Chau Hwang; Ping, Yueh Hsin

doi:10.1117/1.jbo.19.1.011018

Cited by 29 publications

(30 citation statements)

References 45 publications

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“…Here, we report findings that suggest a biphasic response of autophagy to DENV infection, where DENV infection initially activates and then later on inhibits autophagy. Although it has been reported by several groups that multiple components of the autophagy pathway are required for DENV replication (4,6,7,(27)(28)(29), we demonstrate here that DENV activates autophagy only during the early infection stage. Moreover, we established that, by 24 h after infection with the NGC strain, autophagosome formation is inhibited and lysosomal degradation of AVs is reduced.…”

Section: Discussionmentioning

confidence: 70%

“…Autophagic flux is determined by the capacity of the cell to degrade forming autophagosomes, which occurs within minutes (25), and AV content increases upon disruption to lysosomal activity. It is commonly reported that DENV infection increases AV number as detected by high-resolution microscopy (6,7,24,26,27) and Western blot analysis of lipidated LC3-II levels (5,6,28,29). Notably, in autophagosomes colocalizing with lysosomes, pronounced pH-dependent quenching of green fluorescent protein (GFP)-LC3 fluorescence (30) was not observed (7), suggesting that DENV may reduce lysosomal acidification and so disrupt lysosomal function.…”

mentioning

confidence: 99%

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Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Metz

Chiramel

Chatel‐Chaix

et al. 2015

J Virol

103

104

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Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCEAutophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus. Dengue virus (DENV) is a member of the family Flaviviridae and is responsible for one of the most common infections transmitted to humans by mosquitoes. DENV is a positive-st...

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Section: Discussionmentioning

confidence: 70%

mentioning

confidence: 99%

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Metz

Chiramel

Chatel‐Chaix

et al. 2015

J Virol

103

104

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“…Tracking experiments were performed as previously described (34) with minor modifications. In brief, 2 ϫ 10 5 HeLa cells were seeded in a 35-mm glass-bottom culture dish (MatTek, USA) and incubated overnight at 37°C.…”

mentioning

confidence: 99%

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

Chu¹,

Lee

et al. 2015

J Virol

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Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis.We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm. V accinia virus is the prototype of the Orthopoxvirus genus in the family Poxviridae, which includes the variola virus that causes smallpox diseases. It has a broad host range and infects a wide variety of cell lines and animals. Vaccinia virus produces two forms of infectious virions, mature virus (MV) and extracellular virus (EV), which contain different membrane proteins (1). MV particles are abundant in infected cells and contain ϳ80 viral proteins (2, 3), which contribute to the complex virus entry processes that reportedly vary among different cells and virus strains (4-12).In HeLa cells, vaccinia MV initially attaches to cellular surface component glycosaminoglycans (13-15) and the extracellular matrix protein laminin (16). MV particles then cluster at plasma membrane lipid rafts (17) where the virus further interacts with integrin ␤1 (18) and CD98 receptor molecu...

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“…This interaction is required for the inhibition of autophagosome maturation and fusion with the lysosomes. [68][69][70]73 While iLIR could detect several WxxL motifs in TRS1 sequence, a single one in an intrinsically disordered region was identified for ICP34.5 whose sequence (64-RQWLHV-69) is quite well conserved among 4 strains of HSV-1 and one strain of HSV-2. However, to date, there is no evidence of association between ICP34.5 and LC3 proteins.…”

Section: Viruses Inhibiting Autophagymentioning

confidence: 99%

“…Studies have shown that NS1 protein from Dengue virus type 2 (DENV-2) colocalizes with LC3 and that DENV-2 particles and autophagosomes travel together during viral infection. 58,74 In contrast to DENV-2, NS1 protein from DENV-3 displays a low level of colocalization with LC3. 75 Sequence alignment of NS1 proteins from DENV-2 and DENV-3 showed that they are highly conserved.…”

Section: Viruses Activating Autophagymentioning

confidence: 99%

iLIR@viral: A web resource for LIR motif-containing proteins in viruses

et al. 2017

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Macroautophagy/autophagy has been shown to mediate the selective lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. Autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. Atg8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. Such interactions are usually mediated through LC3-interacting region (LIR) motifs. So far, only one viral protein has been experimentally shown to have a functional LIR motif, leaving open a vast field for investigation. Here, we have developed the iLIR@viral database (http://ilir.uk/virus/) as a freely accessible web resource listing all the putative canonical LIR motifs identified in viral proteins. Additionally, we used a curated text-mining analysis of the literature to identify novel putative LIR motif-containing proteins (LIRCPs) in viruses. We anticipate that iLIR@viral will assist with elucidating the full complement of LIRCPs in viruses.

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Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection

Cited by 29 publications

References 45 publications

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

iLIR@viral: A web resource for LIR motif-containing proteins in viruses

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