SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication

ABSTRACTSmall ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular geneUbc9, which encodes the sole E2-conjugating enzyme required for SUMOylation. Byin vivoandin vitroSUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication.IMPORTANCESUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.

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Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo

Journal of General Virology ◽

10.1099/jgv.0.001669 ◽

2021 ◽

Vol 102 (10) ◽

Author(s):

Wisam-Hamzah Al Shujairi ◽

Luke P. Kris ◽

Kylie van der Hoek ◽

Evangeline Cowell ◽

Gustavo Bracho-Granado ◽

...

Keyword(s):

Dengue Virus ◽

Immune Cell ◽

Inflammatory Responses ◽

Denv Infection ◽

Brain Morphology ◽

Moderate Increase ◽

Tnf Α ◽

Significant Difference

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip−/−) showed enhanced DENV infection, accompanied by increased IFN-β and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip−/− mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip−/− compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-β, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip−/− mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip−/− DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-β, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip−/− mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

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The Role of Tissue Oxygen Tension in Dengue Virus Replication

Cells ◽

10.3390/cells7120241 ◽

2018 ◽

Vol 7 (12) ◽

pp. 241 ◽

Cited By ~ 8

Author(s):

Efseveia Frakolaki ◽

Panagiota Kaimou ◽

Maria Moraiti ◽

Katerina Kalliampakou ◽

Kalliopi Karampetsou ◽

...

Keyword(s):

Dengue Virus ◽

Oxygen Tension ◽

Atmospheric Oxygen ◽

Rna Replication ◽

Metabolic Reprogramming ◽

Anaerobic Glycolysis ◽

Low Oxygen ◽

Hypoxia Response

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.

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Preclinical Profile and Clinical Efficacy of a Novel Hepatitis C Virus NS5A Inhibitor, EDP-239

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00808-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6207-6215 ◽

Cited By ~ 3

Author(s):

Christopher M. Owens ◽

Bradley B. Brasher ◽

Alex Polemeropoulos ◽

Michael H. J. Rhodin ◽

Nicole McAllister ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Viral Rna ◽

Controlled Clinical Trial ◽

Genotype 1A ◽

Pharmacokinetic Properties ◽

Direct Acting

ABSTRACTEDP-239, a novel hepatitis C virus (HCV) inhibitor targeting nonstructural protein 5A (NS5A), has been investigatedin vitroandin vivo. EDP-239 is a potent, selective inhibitor with potency at picomolar to nanomolar concentrations against HCV genotypes 1 through 6. In the presence of human serum, the potency of EDP-239 was reduced by less than 4-fold. EDP-239 is additive to synergistic with other direct-acting antivirals (DAAs) or host-targeted antivirals (HTAs) in blocking HCV replication and suppresses the selection of resistancein vitro. Furthermore, EDP-239 retains potency against known DAA- or HTA-resistant variants, with half-maximal effective concentrations (EC50s) equivalent to those for the wild type. In a phase I, single-ascending-dose, placebo-controlled clinical trial, EDP-239 demonstrated excellent pharmacokinetic properties that supported once daily dosing. A single 100-mg dose of EDP-239 resulted in reductions in HCV genotype 1a viral RNA of >3 log10IU/ml within the first 48 h after dosing and reductions in genotype 1b viral RNA of >4-log10IU/ml within 96 h. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.)

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Epitope Determinants of a Chimpanzee Dengue Virus Type 4 (DENV-4)-Neutralizing Antibody and Protection against DENV-4 Challenge in Mice and Rhesus Monkeys by Passively Transferred Humanized Antibody

Journal of Virology ◽

10.1128/jvi.01420-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12766-12774 ◽

Cited By ~ 54

Author(s):

Ching-Juh Lai ◽

Ana P. Goncalvez ◽

Ruhe Men ◽

Claire Wernly ◽

Olivia Donau ◽

...

Keyword(s):

Dengue Virus ◽

Virus Type ◽

High Titer ◽

Neutralizing Antibody ◽

Denv Infection ◽

Passive Transfer ◽

Escape Mutant ◽

Protective Capacity

ABSTRACT The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ΔD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ΔD at 20 μg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ΔD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 106 MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.

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Discovery of Dengue Virus NS4B Inhibitors

Journal of Virology ◽

10.1128/jvi.00855-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8233-8244 ◽

Cited By ~ 51

Author(s):

Qing-Yin Wang ◽

Hongping Dong ◽

Bin Zou ◽

Ratna Karuna ◽

Kah Fei Wan ◽

...

Keyword(s):

Amino Acid ◽

Dengue Virus ◽

Transmembrane Protein ◽

Selective Inhibition ◽

Denv Infection ◽

Viral Pathogens ◽

Replication Complex ◽

First Time

ABSTRACTThe four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV bothin vitroandin vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has goodin vivopharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients.IMPORTANCEDengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) bothin vitroandin vivo. Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

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Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

Journal of Virology ◽

10.1128/jvi.79.11.6631-6643.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6631-6643 ◽

Cited By ~ 238

Author(s):

Diego E. Alvarez ◽

María F. Lodeiro ◽

Silvio J. Ludueña ◽

Lía I. Pietrasanta ◽

Andrea V. Gamarnik

Keyword(s):

Dengue Virus ◽

Rna Binding ◽

Rna Replication ◽

Virus Genome ◽

Viral Rna ◽

Physical Interaction ◽

Virus Rna ◽

Rna Elements ◽

Rna Interaction

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

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Differential replication of dengue virus serotypes 2 and 3 in coinfections of C6/36 cells and Aedes aegypti mosquitoes

The Journal of Infection in Developing Countries ◽

10.3855/jidc.3978 ◽

2014 ◽

Vol 8 (07) ◽

pp. 876-884 ◽

Cited By ~ 8

Author(s):

Diana Carolina Quintero-Gil ◽

Marta Ospina ◽

Jorge Emilio Osorio-Benitez ◽

Marlen Martinez-Gutierrez

Keyword(s):

Aedes Aegypti ◽

Dengue Virus ◽

Cell Viability ◽

Denv Infection ◽

Replication Capacity ◽

Denv Serotypes ◽

Viral Genomes ◽

Diphenyltetrazolium Bromide

Introduction: Different dengue virus (DENV) serotypes have been associated with greater epidemic potential. In turn, the increased frequency in cases of severe forms of dengue has been associated with the cocirculation of several serotypes. Because Colombia is a country with an endemic presence of all four DENV serotypes, the aim of this study was to evaluate the in vivo and in vitro replication of the DENV-2 and DENV-3 strains under individual infection and coinfection conditions. Methodology: C6/36HT cells were infected with the two strains individually or simultaneously (coinfection). Replication capacity was evaluated by RT-qPCR, and the effects on cell viability were assessed with an MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Additionally, Aedes aegypti mosquitoes were artificially fed the two strains of each serotype individually or simultaneously. The viral genomes were quantified by RT-qPCR and the survival of the infected mosquitoes was compared to that of uninfected controls. Results: In single infections, three strains significantly affected C6/36HT cell viability, but no significant differences were found in the replication capacities of the strains of the same serotype. In the in vivo infections, mosquito survival was not affected, and no significant differences in replication between strains of the same serotype were found. Finally, in coinfections, serotype 2 replicated with a thousandfold greater efficiency than serotype 3 did both in vitro and in vivo. Conclusions: Due to the cocirculation of serotypes in endemic regions, further studies of coinfections in a natural environment would further an understanding of the transmission dynamics that affect DENV infection epidemiology.

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Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription

Journal of Virology ◽

10.1128/jvi.77.9.5098-5108.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5098-5108 ◽

Cited By ~ 33

Author(s):

Pablo Gastaminza ◽

Beatriz Perales ◽

Ana M. Falcón ◽

Juan Ortín

Keyword(s):

Influenza Virus ◽

Rna Replication ◽

Terminal Region ◽

Viral Rna ◽

Mrna Accumulation ◽

Virus Rna ◽

Transcription In Vitro ◽

Type Mutant

ABSTRACT PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

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Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo

Scientific Reports ◽

10.1038/s41598-020-78639-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Takeshi Kurosu ◽

Keiko Hanabara ◽

Azusa Asai ◽

Sabar Pambudi ◽

Supranee Phanthanawiboon ◽

...

Keyword(s):

Dengue Virus ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Evaluation Process ◽

Denv Infection ◽

Interferon Α ◽

Double Knockout ◽

Virus Envelope

AbstractIn a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/β–γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

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