Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication

2013 ◽  
Vol 99 (2) ◽  
pp. 165-171 ◽  
Author(s):  
Koen W.R. van Cleef ◽  
Gijs J. Overheul ◽  
Michael C. Thomassen ◽  
Suzanne J.F. Kaptein ◽  
Andrew D. Davidson ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Replication ◽  
Genomic Rna ◽  
Virus Inhibitor ◽  
Ns4b Protein

scholarly journals Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

2015 ◽  
Vol 89 (13) ◽  
pp. 6746-6760 ◽  
Author(s):  
Nenavath Gopal Naik ◽  
Huey-Nan Wu
Keyword(s):  
Life Cycle ◽  
Recombinant Protein ◽  
Dengue Virus ◽  
Protein Expression ◽  
Nonstructural Protein ◽  
Recombinant Protein Expression ◽  
Rna Replication ◽  
Viral Production ◽  
Glycosylation Sites ◽  
Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

scholarly journals A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

2015 ◽  
Vol 89 (14) ◽  
pp. 7170-7186 ◽  
Author(s):  
Laurent Chatel-Chaix ◽  
Wolfgang Fischl ◽  
Pietro Scaturro ◽  
Mirko Cortese ◽  
Stephanie Kallis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Virus Replication ◽  
Strong Evidence ◽  
Drug Target ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Replication Cycle ◽  
Infected Cells ◽  
Ns3 Protease

ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

scholarly journals Human MxB Inhibits the Replication of Hepatitis C Virus

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Dong-Rong Yi ◽  
Ni An ◽  
Zhen-Long Liu ◽  
Feng-Wen Xu ◽  
Kavita Raniga ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dengue Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Rna Replication ◽  
Encephalitis Virus ◽  
Common Mechanism ◽  
Type I ◽  
Dependent Manner

ABSTRACTType I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of theFlaviviridaefamily, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.IMPORTANCEViruses of theFlaviviridaefamily cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of theFlaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

scholarly journals The Large Delta Antigen of Hepatitis Delta Virus Potently Inhibits Genomic but Not Antigenomic RNA Synthesis: a Mechanism Enabling Initiation of Viral Replication

2000 ◽  
Vol 74 (16) ◽  
pp. 7375-7380 ◽  
Author(s):  
Lucy E. Modahl ◽  
Michael M. C. Lai
Keyword(s):  
Life Cycle ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Dominant Negative ◽  
Genomic Rna ◽  
Inhibitory Effects ◽  
Hepatitis Delta ◽  
Artificial Dna ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.

scholarly journals The Role of Tissue Oxygen Tension in Dengue Virus Replication

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 241 ◽  
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.

Stachyonic Acid: A Dengue Virus Inhibitor from Basilicum polystachyon

2019 ◽  
Vol 25 (22) ◽  
pp. 5664-5667 ◽  
Author(s):  
Yuen P. Tan ◽  
Sevan D. Houston ◽  
Naphak Modhiran ◽  
Andrei I. Savchenko ◽  
Glen M. Boyle ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Inhibitor

scholarly journals Translation-coupled RNA replication and parasitic replicators in membrane-free compartments

2020 ◽  
Vol 56 (87) ◽  
pp. 13453-13456
Author(s):  
Ryo Mizuuchi ◽  
Norikazu Ichihashi
Keyword(s):  
Liquid Phase ◽  
Rna Replication ◽  
Genomic Rna ◽  
Self Replication

Liquid–liquid phase-separated droplets concentrated a genomic RNA and translation proteins to activate and support RNA self-replication by its encoded protein.

scholarly journals Scanning Mutagenesis Studies Reveal a Potential Intramolecular Interaction within the C-Terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly and Secretion

2015 ◽  
Vol 89 (8) ◽  
pp. 4281-4295 ◽  
Author(s):  
Ren-Huang Wu ◽  
Ming-Han Tsai ◽  
Day-Yu Chao ◽  
Andrew Yueh
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Virus Assembly ◽  
Intramolecular Interaction ◽  
Rna Replication ◽  
The Other ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Virus Life Cycle

ABSTRACTThe NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transienttrans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated.IMPORTANCEThe reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

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