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

2015 ◽  
Vol 89 (15) ◽  
pp. 8026-8041 ◽  
Author(s):  
Philippe Metz ◽  
Abhilash Chiramel ◽  
Laurent Chatel-Chaix ◽  
Gualtiero Alvisi ◽  
Peter Bankhead ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dengue Virus ◽  
Viral Replication ◽  
Normal Growth ◽  
Proteasomal Degradation ◽  
Denv Infection ◽  
Replication Cycle ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Initial Activation

ABSTRACTAutophagic 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.

scholarly journals Dengue Virus Activates the AMP Kinase-mTOR Axis To Stimulate a Proviral Lipophagy

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Tristan X. Jordan ◽  
Glenn Randall
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Lipid Droplets ◽  
Adenosine Monophosphate ◽  
Denv Infection ◽  
Ampk Signaling ◽  
Selective Autophagy ◽  
Dependent Inactivation ◽  
Mtorc1 Signaling ◽  
Infected Cells

ABSTRACT Robust dengue virus (DENV) replication requires lipophagy, a selective autophagy that targets lipid droplets. The autophagic mobilization of lipids leads to increased β-oxidation in DENV-infected cells. The mechanism by which DENV induces lipophagy is unknown. Here, we show that infection with DENV activates the metabolic regulator 5′ adenosine-monophosphate activated kinase (AMPK), and that the silencing or pharmacological inhibition of AMPK activity decreases DENV replication and the induction of lipophagy. The activity of the mechanistic target of rapamycin complex 1 (mTORC1) decreases in DENV-infected cells and is inversely correlated with lipophagy induction. Constitutive activation of mTORC1 by depletion of tuberous sclerosis complex 2 (TSC2) inhibits lipophagy induction in DENV-infected cells and decreases viral replication. While AMPK normally stimulates TSC2-dependent inactivation of mTORC1 signaling, mTORC1 inactivation is independent of AMPK activation during DENV infection. Thus, DENV stimulates and requires AMPK signaling as well as AMPK-independent suppression of mTORC1 activity for proviral lipophagy. IMPORTANCE Dengue virus alters host cell lipid metabolism to promote its infection. One mechanism for altered metabolism is the induction of a selective autophagy that targets lipid droplets, termed lipophagy. Lipophagy mobilizes lipid stores, resulting in enhanced β-oxidation and viral replication. We show here that DENV infection activates and requires the central metabolic regulator AMPK for its replication and the induction of lipophagy. This is required for the induction of lipophagy, but not basal autophagy, in DENV-infected cells.

scholarly journals The ULK1-FBXW5-SEC23B nexus controls autophagy

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yeon-Tae Jeong ◽  
Daniele Simoneschi ◽  
Sarah Keegan ◽  
David Melville ◽  
Natalia S Adler ◽  
...  
Keyword(s):  
Coat Protein ◽  
Protein Complex ◽  
Proteasomal Degradation ◽  
Nutrient Starvation ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Complex Ii ◽  
Intermediate Compartment ◽  
F Box Protein ◽  
Efficient Execution

In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

scholarly journals The dengue virus non-structural protein 1 (NS1) interacts with the putative epigenetic regulator DIDO1 to promote flavivirus replication.

2021 ◽  
Author(s):  
gerson caraballo ◽  
Romel Rosales ◽  
Mercedes Viettri ◽  
Siyuan Ding ◽  
Harry B Greenberg ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Ribosomal Proteins ◽  
Denv Infection ◽  
Structural Protein ◽  
Transcription Analysis ◽  
Multifunctional Protein ◽  
Rna Surveillance ◽  
Mosquito Cells ◽  
Expression Pathways

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein.

scholarly journals The ULK1-FBXW5-SEC23B nexus controls autophagy

2018 ◽  
Author(s):  
Yeon-Tae Jeong ◽  
Daniele Simoneschi ◽  
Sarah Keegan ◽  
David Melville ◽  
Natalia S. Adler ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Proteasomal Degradation ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Intermediate Compartment ◽  
F Box Protein

ABSTRACTIn response to nutrient deprivation, the cell needs to mobilize an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and a source for LC3 lipidation, COPII (Coat Protein Complex II) localizes to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and promotes autophagosome biogenesis. However, the molecular mechanisms that, in response to starvation, divert COPII from the secretory pathway to the autophagic pathway are largely unknown. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting its degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ERGIC, promoting autophagic flux. Induction of autophagy and localization of both SEC23B and SEC24B to the ERGIC in response to nutrient deprivation are significantly reduced in SEC23B(S186A) knock-in cells. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

scholarly journals Evaluation of AT-752, a double prodrug of a guanosine nucleotide analog with in vitro and in vivo activity against dengue and other flaviviruses

2021 ◽  
Author(s):  
Steven S. Good ◽  
Ashleigh Shannon ◽  
Kai Lin ◽  
Adel Moussa ◽  
Justin G. Julander ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Mononuclear Cells ◽  
Potent Inhibitor ◽  
Denv Infection ◽  
Peripheral Blood Mononuclear ◽  
Denv Serotypes ◽  
Nucleotide Analog

Every year millions of people worldwide are infected with dengue virus (DENV), with a significant number developing severe life-threatening disease. There are currently no broadly indicated vaccines or therapeutics available for treatment of DENV infection. Here, we show that AT-281, the free base of AT-752, an orally available double prodrug of a guanosine nucleotide analog, was a potent inhibitor of DENV serotypes 2 and 3 in vitro , requiring concentrations of 0.48 and 0.77 μM, respectively, to inhibit viral replication by 50% (EC 50 ) in Huh-7 cells. AT-281 was also a potent inhibitor of all other flaviviruses tested with EC 50 values ranging from 0.19 to 1.41 μM. Little to no cytotoxicity was observed for AT-281 at concentrations up to 170 μM. After oral administration of AT-752, substantial levels of the active triphosphate metabolite AT-9010 were formed in vivo in peripheral blood mononuclear cells of mice, rats and monkeys. Furthermore, AT-9010 competed with guanosine triphosphate in RNA template-primer elongation assays with DENV-2 RNA polymerase, which is essential for viral replication, with incorporation of AT-9010 resulting in termination of RNA synthesis. In AG129 mice infected with DENV D2Y98P, treatment with AT-752 significantly reduced viremia and morbidity and increased survival. The demonstrated in vitro and in vivo activity of AT-752 suggest that it is a promising compound for the treatment of dengue virus infection, and is currently under evaluation in clinical studies.

scholarly journals Dengue Virus Induces and Requires Glycolysis for Optimal Replication

2014 ◽  
Vol 89 (4) ◽  
pp. 2358-2366 ◽  
Author(s):  
Krystal A. Fontaine ◽  
Erica L. Sanchez ◽  
Roman Camarda ◽  
Michael Lagunoff
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Building Blocks ◽  
Denv Infection ◽  
Glycolytic Pathway ◽  
Metabolic Inhibitors ◽  
Human Pathogens ◽  
Glucose Transporter 1 ◽  
Infected Cells ◽  
The Impact

ABSTRACTViruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of theFlaviviridaefamily, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future.IMPORTANCEApproximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens.

scholarly journals Oligonucleotide-Based Approaches to Inhibit Dengue Virus Replication

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 956
Author(s):  
Kingshuk Panda ◽  
Kalichamy Alagarasu ◽  
Deepti Parashar
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Viral Infections ◽  
Public Health Problem ◽  
Denv Infection ◽  
Viral Rna ◽  
Effective Vaccine ◽  
Cellular Processes ◽  
Antiviral Therapies ◽  
Life Threatening

Dengue fever is one of the most common viral infections affecting humans. It is an expanding public health problem, particularly in tropical and subtropical regions. No effective vaccine or antiviral therapies against Dengue virus (DENV) infection are available. Therefore, there is a strong need to develop safe and effective therapeutic strategies that can reduce the burden and duration of hospitalizations due to this life-threatening disease. Oligonucleotide-based strategies are considered as an attractive means of inhibiting viral replication since oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The resultant targeted destruction of viral RNA interferes with viral replication without inducing any adverse effects on cellular processes. In this review, we elaborate the ribozymes, RNA interference, CRISPR, aptamer and morpholino strategies for the inhibition of DENV replication and discuss the challenges involved in utilizing such approaches.

An experimental and theoretical approach to understand Fever, DENF & its cure

2020 ◽  
Vol 20 ◽  
Author(s):  
Vijay Kumar Vishvakarma ◽  
Ramesh Chandra ◽  
Prashant Singh
Keyword(s):  
Catalytic Activity ◽  
Dengue Virus ◽  
Theoretical Approach ◽  
Major Part ◽  
Genomic Structure ◽  
Denv Infection ◽  
Structural Proteins ◽  
Experimental Approaches ◽  
Ns3 Protease ◽  
Prime Target

: Fever is a response of human body due to an increase the temperature against the certain stimuli. It may be associated with several reasons and one of the major causes of fever is mosquito bite. Fever due to dengue virus (DENV) infection is being paid most attention out of several other fevers because of a large number of deaths reported worldwide. Dengue virus is transmitted by biting of the mosquitoes, Aedes aegypti and Aedes albopictus. DENV1, DENV2, DENV3 and DENV4 are the four serotypes of dengue virus and these serotypes have 65% similarities in their genomic structure. Genome of DENV is composed of single stranded RNA and it encodes for the polyprotein. Structural and non-structural proteins (nsP) are the two major part of protese. Researchers have paid high attention on the non-structural protease (nsP) of DENV like nsP1, nsP2A, nsP2B, nsP3, nsP4A, nsP4B and nsP5. The NS2B-NS3 protease of DENV is the prime target of the researchers as it is responsible for the catalytic activity. In the present time, Dengvaxia (vaccine) is being recommended to the patients suffering severely due to DENV infection in few countries only. Till date, neither a vaccine nor an effective medicine is available to combat with all four serotypes. This review describes the fever, its causes and studies to cure the infection due to DENV using theoretical and experimental approaches.

scholarly journals Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1393
Author(s):  
Thanyaporn Dechtawewat ◽  
Sittiruk Roytrakul ◽  
Yodying Yingchutrakul ◽  
Sawanya Charoenlappanit ◽  
Bunpote Siridechadilok ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
Multifunctional Protein ◽  
Nonstructural Protein 1 ◽  
Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

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