Investigating Dengue Virus Nonstructural Protein 5 (NS5) Nuclear Import

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

Journal of Virology ◽

10.1128/jvi.00423-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6746-6760 ◽

Cited By ~ 26

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.

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

Journal of Virology ◽

10.1128/jvi.00867-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7170-7186 ◽

Cited By ~ 26

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.

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Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay

Journal of Medical Virology ◽

10.1002/jmv.23204 ◽

2012 ◽

Vol 84 (3) ◽

pp. 490-499 ◽

Cited By ~ 13

Author(s):

Marine Steidel ◽

Romain Fragnoud ◽

Michelle Guillotte ◽

Céline Roesch ◽

Sandrine Michel ◽

...

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Immunodominant Epitope ◽

Infected Material ◽

Tof Ms

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High Circulating Levels of the Dengue Virus Nonstructural Protein NS1 Early in Dengue Illness Correlate with the Development of Dengue Hemorrhagic Fever

The Journal of Infectious Diseases ◽

10.1086/343813 ◽

2002 ◽

Vol 186 (8) ◽

pp. 1165-1168 ◽

Cited By ~ 408

Author(s):

Daniel H. Libraty ◽

Paul R. Young ◽

Darren Pickering ◽

Timothy P. Endy ◽

Siripen Kalayanarooj ◽

...

Keyword(s):

Dengue Virus ◽

Dengue Hemorrhagic Fever ◽

Nonstructural Protein ◽

Hemorrhagic Fever ◽

Circulating Levels

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Establishment and characterization of dengue virus type 2 nonstructural protein 1 specific T cell lines

Comparative Immunology Microbiology and Infectious Diseases ◽

10.1016/j.cimid.2010.01.002 ◽

2010 ◽

Vol 33 (6) ◽

pp. e75-e80 ◽

Cited By ~ 1

Author(s):

Yang Xiao-meng ◽

Jiang Li-fang ◽

Tang Yun-xia ◽

Yin Yue ◽

Liu Wen-quan ◽

...

Keyword(s):

T Cell ◽

Dengue Virus ◽

Cell Lines ◽

Virus Type ◽

Nonstructural Protein ◽

Nonstructural Protein 1 ◽

T Cell Lines ◽

Dengue Virus Type 2

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Modification ofS-Adenosyl-l-Homocysteine as Inhibitor of Nonstructural Protein 5 Methyltransferase Dengue Virus Through Molecular Docking and Molecular Dynamics Simulation

Drug Target Insights ◽

10.1177/1177392817701726 ◽

2017 ◽

Vol 11 ◽

pp. 117739281770172 ◽

Cited By ~ 12

Author(s):

Usman Sumo Friend Tambunan ◽

Mochammad Arfin Fardiansyah Nasution ◽

Fauziah Azhima ◽

Arli Aditya Parikesit ◽

Erwin Prasetya Toepak ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Docking ◽

Molecular Dynamics Simulation ◽

Dengue Virus ◽

Nonstructural Protein ◽

Dynamics Simulation

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NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models

Science Translational Medicine ◽

10.1126/scitranslmed.abb2181 ◽

2021 ◽

Vol 13 (579) ◽

pp. eabb2181

Author(s):

Stephanie A. Moquin ◽

Oliver Simon ◽

Ratna Karuna ◽

Suresh B. Lakshminarayana ◽

Fumiaki Yokokawa ◽

...

Keyword(s):

Dengue Virus ◽

Nonstructural Protein ◽

Antiviral Drug ◽

Pharmacokinetic Studies ◽

Resistance Mutations ◽

Binding Studies ◽

Log Reduction ◽

Elimination Half ◽

Preclinical Animal Models

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

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