Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B*

Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH2-terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle.

• Publication year

  2006

• Venue

  Journal of Biological Chemistry

• Publication date

  2006-03-31

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1074/jbc.M512697200PMID 16436383
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Mutational Analysis of Hepatitis C Virus Nonstructural Protein 5A: Potential Role of Differential Phosphorylation in RNA Replication and Identification of a Genetically Flexible Domain

  2005cited by this paper
• Flavivirus infection: essential ultrastructural changes and association of Kunjin virus NS3 protein with microtubules

  2005cited by this paper
• Inhibition of Interferon Signaling by the New York 99 Strain and Kunjin Subtype of West Nile Virus Involves Blockage of STAT1 and STAT2 Activation by Nonstructural Proteins

  2005cited by this paper
• Immunofluorescent sites in vero cells infected with the flavivirus Kunjin

  2005cited by this paper
• Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA

  2005cited by this paper
• Inhibition of Alpha/Beta Interferon Signaling by the NS4B Protein of Flaviviruses

  2005cited by this paper
• West Nile Virus Inhibits the Signal Transduction Pathway of Alpha Interferon

  2005cited by this paper
• An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

  2004cited by this paper
• A Structural Basis for the Inhibition of the NS5 Dengue Virus mRNA 2′-O-Methyltransferase Domain by Ribavirin 5′-Triphosphate*

  2004cited by this paper
• A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

  2004cited by this paper
• Comparisons of physical separation methods of Kunjin virus-induced membranes.

  2004cited by this paper
• A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4.

  2003cited by this paper
• Inhibition of interferon signaling by dengue virus

  2003cited by this paper
• Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

  2003cited by this paper
• Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells.

  2003cited by this paper
• Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

  2002cited by this paper
• Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein*

  2002cited by this paper
• Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo

  2002cited by this paper
• Inhibition of protein synthesis by the nonstructural proteins NS4A and NS4B of hepatitis C virus.

  2002cited by this paper
• Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century.

  2002cited by this paper
• An RNA cap (nucleoside‐2′‐O‐)‐methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization

  2002cited by this paper
• Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented intrans

  2001cited by this paper
• The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein.

  2001cited by this paper
• Molecular Cloning: A Laboratory Manual

  2001cited by this paper
• Organization of Two Transmissible Gastroenteritis Coronavirus Membrane Protein Topologies within the Virion and Core

  2001cited by this paper
• Dual Topology of the Hepatitis B Virus Large Envelope Protein

  2001cited by this paper
• Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

  2001cited by this paper
• Hepatitis C virus nonstructural protein NS4B transforms NIH3T3 cells in cooperation with the Ha-ras oncogene.

  2000cited by this paper
• cis- and trans-Acting Elements in Flavivirus RNA Replication

  2000cited by this paper
• Genetic Interaction of Flavivirus Nonstructural Proteins NS1 and NS4A as a Determinant of Replicase Function

  1999cited by this paper
• Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A.

  1998cited by this paper
• Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus.

  1997cited by this paper
• Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures

  1997cited by this paper
• Proteolytic activation of tick-borne encephalitis virus by furin

  1997cited by this paper
• trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication

  1997cited by this paper
• Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication.

  1996cited by this paper
• Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

  1996cited by this paper
• Membrane association of influenza virus matrix protein does not require specific hydrophobic domains or the viral glycoproteins.

  1996cited by this paper
• Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum

  1995cited by this paper
• Novel transmembrane topology of the hepatitis B virus envelope proteins.

  1995cited by this paper
• Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion

  1995cited by this paper
• Association between NS3 and NS5 Proteins of Dengue Virus Type 2 in the Putative RNA Replicase Is Linked to Differential Phosphorylation of NS5 (*)

  1995cited by this paper
• A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

  1994cited by this paper
• Post‐translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.

  1994cited by this paper
• NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies

  1994cited by this paper
• Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome.

  1993cited by this paper
• Processing of the dengue virus type 2 proteins prM and C-prM.

  1993cited by this paper
• Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site

  1993cited by this paper
• The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity.

  1993cited by this paper
• Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin.

  1992cited by this paper
• Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease

  1992cited by this paper
• Monoclonal antibodies identify the NS5 yellow fever virus non-structural protein in the nuclei of infected cells.

  1992cited by this paper
• Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins

  1991cited by this paper
• Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo.

  1991cited by this paper
• New mammalian expression vectors

  1990cited by this paper
• Flavivirus genome organization, expression, and replication.

  1990cited by this paper
• Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein.

  1990cited by this paper
• Product review. New mammalian expression vectors.

  1990cited by this paper
• N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases.

  1989cited by this paper
• Yellow fever virus proteins NS2A, NS2B, and NS4B: identification and partial N-terminal amino acid sequence analysis.

  1989cited by this paper
• Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.

  1982cited by this paper
• Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum

  1982cited by this paper
• Sequential events in the morphogenesis of japanese Encephalitis virus.

  1980cited by this paper

• Membrane Remodeling Activity of Dengue Virus NS4A and NS1 Proteins: A Computational Perspective

  2026cites this paper
• In vitro synthesis of active recombinant orthoflavivirus nonstructural polyproteins in detergent micelles for biochemical analysis

  2026cites this paper
• Zika NS2B Protein: In Vitro Formation of Large Multimeric Networks

  2026cites this paper
• Replication differences of SARS-CoV-2 lineages may arise from unique RNA replication characteristics and nucleocapsid protein expression

  2025cites this paper
• AlphaFold Prediction of Protein–Protein Interactions in the Flaviviridae Proteomes

  2025cites this paper
• Dengue virus-host interactions: Structural and mechanistic insights for future therapeutic strategies.

  2025cites this paper
• Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation

  2025cites this paper
• Cellular Biology: Virus-Host Interactions and RNA Structure and Function.

  2025cites this paper
• Flavivirus NS4B proteins do not form homodimers: discrepancies with an AlphaFold-based oligomeric model

  2025cites this paper
• Development of New Live-Attenuated Vaccine Candidates Lacking Antibody-Dependent Enhancement (ADE) Against Dengue

  2025cites this paper
• Discovery of a potent anti-Zika virus benzamide series targeting the viral protein NS4B

  2025cites this paper
• Knowledge-based mitigation of the environmental risk of Orthoflavivirus live-attenuated vaccines by targeting viral encoded determinants for the mosquito attenuated phenotype.

  2025cites this paper
• Functional Roles and Host Interactions of Orthoflavivirus Non-Structural Proteins During Replication

  2025cites this paper
• The ACBD3 protein coordinates ER-Golgi contacts to enable productive TBEV infection

  2025cites this paper
• Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation

  2025cites this paper
• Dengue and Zika virus NS4B proteins differ in topology and in determinants of ER membrane protein complex dependency

  2024cites this paper
• Characterization of duck tembusu virus NS2A membrane topology and functional residues in transmembrane domain-3 on viral proliferation

  2024cites this paper
• Mechanistic insights into dengue virus inhibition by a clinical trial compound NITD-688

  2024cites this paper
• The flavivirus protein NS4B recruits the cis-Golgi protein ACBD3 to modify ER-Golgi trafficking for virion release

  2024cites this paper
• Advancements in Research on Duck Tembusu Virus Infections

  2024cites this paper
• The topological model of NS4B and its TMD3 in duck TMUV proliferation

  2024influential citation
• Understanding Dengue Proteins: Key Players in Pathogenesis and Treatment Strategies

  2024cites this paper
• Advances in drug discovery of flavivirus NS2B-NS3pro serine protease inhibitors for the treatment of Dengue, Zika, and West Nile viruses.

  2024cites this paper
• 1H, 15N and 13C backbone resonance assignment of the N-terminal region of Zika virus NS4B protein in detergent micelles

  2024cites this paper
• Molecular epidemiology and pathogenicity of Wesselsbron virus circulating in Africa

  2024cites this paper
• Orthoflaviviral Inhibitors in Clinical Trials, Preclinical In Vivo Efficacy Targeting NS2B-NS3 and Cellular Antiviral Activity via Competitive Protease Inhibition

  2024cites this paper
• Unraveling the Genomic Evolution of Dengue Virus Serotype 1: A Case Study from Yantai, China

  2024cites this paper
• In silico homology modeling of dengue virus non-structural 4B (NS4B) protein and its molecular docking studies using triterpenoids

  2024cites this paper
• Pan-serotype dengue virus inhibitor JNJ-A07 targets NS4A-2K-NS4B interaction with NS2B/NS3 and blocks replication organelle formation

  2024cites this paper
• Multienzyme-like active MnO2 nanozyme with ROS scavenging for inflammatory injury therapy induced by avian flavivirus through antiviral function.

  2024cites this paper
• Membrane Retention of West Nile Virus NS5 Depends on NS1 or NS3 for Enzymatic Activity

  2024cites this paper
• Impact of Annexin A2 on virus life cycles

  2024cites this paper
• The endoplasmic reticulum (ER): a crucial cellular hub in flavivirus infection and potential target site for antiviral interventions

  2024cites this paper
• Flavivirus Concentrates Host ER in Main Replication Compartments to Facilitate Replication

  2023cites this paper
• Zika virus NS4B protein targets TANK-binding kinase 1 and inhibits type I interferon production.

  2023cites this paper
• Development in the Inhibition of Dengue Proteases as Drug Targets.

  2023cites this paper
• Roles of NS1 Protein in Flavivirus Pathogenesis.

  2023cites this paper
• Dengue virus NS1 secretion is regulated via importin-subunit β1 controlling expression of the chaperone GRp78 and targeted by the clinical drug ivermectin

  2023cites this paper
• Morphologic and Genetic Characterization of Ilheus Virus, a Potential Emergent Flavivirus in the Americas

  2023cites this paper
• Blocking NS3–NS4B interaction inhibits dengue virus in non-human primates

  2023cites this paper
• A yellow fever virus NS4B inhibitor not only suppresses viral replication, but also enhances the virus activation of RIG-I-like receptor-mediated innate immune response

  2022cites this paper
• Structures and Dynamics of Dengue Virus Nonstructural Membrane Proteins

  2022cites this paper
• Dengue Virus NS4b N-Terminus Disordered Region Interacts with NS3 Helicase C-Terminal Subdomain to Enhance Helicase Activity

  2022cites this paper
• Inhibitory Potential of Chromene Derivatives on Structural and Non-Structural Proteins of Dengue Virus

  2022cites this paper
• Modulation of cellular machineries by Zika virus‐encoded proteins

  2022cites this paper
• An overview on small molecules acting as broad-spectrum agents for yellow fever infection

  2022cites this paper
• Dengue virus NS4B protein as a target for developing antivirals

  2022influential citation
• Flavivirus NS4B protein: Structure, function, and antiviral discovery.

  2022cites this paper
• The Role of the Flavivirus Replicase in Viral Diversity and Adaptation

  2022cites this paper
• Investigating the conformational dynamics of Zika virus NS4B protein

  2021influential citation
• A pan-serotype dengue virus inhibitor targeting the NS3–NS4B interaction

  2021cites this paper
• Roles of Non-Structural Protein 4A in Flavivirus Infection

  2021cites this paper
• Morphological Diversity and Dynamics of Dengue Virus Affecting Antigenicity

  2021cites this paper
• Roles of the Endogenous Lunapark Protein during Flavivirus Replication

  2021cites this paper
• Membrane-Associated Flavivirus Replication Complex—Its Organization and Regulation

  2021cites this paper
• Dengue Virus: Epidemiology, Biology and Disease Aetiology.

  2021cites this paper
• A Novel Series of Indole Alkaloid Derivatives Inhibit Dengue and Zika Virus Infection by Interference with the Viral Replication Complex

  2021cites this paper
• Amelioration of Beta Interferon Inhibition by NS4B Contributes to Attenuating Tembusu Virus Virulence in Ducks

  2021cites this paper
• The Biogenesis of Dengue Virus Replication Organelles Requires the ATPase Activity of Valosin-Containing Protein

  2021cites this paper
• Amaryllidaceae Alkaloid Cherylline Inhibits the Replication of Dengue and Zika Viruses

  2021cites this paper
• Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication

  2021cites this paper
• Novel insights into the function of an N-terminal region of DENV2 NS4B for the optimal helicase activity of NS3.

  2021cites this paper
• NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models

  2021cites this paper
• Compartmentalized replication organelle of flavivirus at the ER and the factors involved

  2021cites this paper
• Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes

  2021cites this paper
• Targeting the protease of West Nile virus.

  2021cites this paper
• Valosin‐containing protein ATPase activity regulates the morphogenesis of Zika virus replication organelles and virus‐induced cell death

  2021cites this paper
• Molecular Insights into the Flavivirus Replication Complex

  2021cites this paper
• Structural and Biochemical Insights Into Flavivirus Proteins.

  2021cites this paper
• Latest Updates in Dengue Fever Therapeutics: Natural, Marine, and Synthetic Drugs.

  2020cites this paper
• A systematic review on phytochemicals for the treatment of dengue

  2020cites this paper
• Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus

  2020cites this paper
• Tick-Borne Encephalitis Virus Adaptation in Different Host Environments and Existence of Quasispecies

  2020cites this paper
• International Journal of Current Microbiology and Applied Sciences

  2020cites this paper
• The entanglement between flaviviruses and ER-shaping proteins

  2020cites this paper
• A role of unfolded protein response in the cellular response to flavivirus infection

  2020cites this paper
• A Non-Replicative Role of the 3′ Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation

  2020cites this paper
• Selective Reactivity of Anti-Japanese Encephalitis Virus NS4B Antibody Towards Different Flaviviruses

  2020cites this paper
• Natural Products with Potential to Treat RNA Virus Pathogens Including SARS-CoV-2

  2020cites this paper
• Replication-Independent Generation and Morphological Analysis of Flavivirus Replication Organelles

  2020cites this paper
• Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication

  2019cites this paper
• The major oligosaccharyl transferase complex genes are not involved in dengue virus replication in Aedes aegypti mosquitoes

  2019cites this paper
• Dengue Virus

  2019cites this paper
• Reciprocal Effects of Fibroblast Growth Factor Receptor Signaling on Dengue Virus Replication and Virion Production.

  2019cites this paper
• The Effects of Autophagy on the Replication of Zika Virus

  2019cites this paper
• A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle

  2019cites this paper
• The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

  2019cites this paper
• Exploiting the unique features of Zika and Dengue proteases for inhibitor design.

  2019cites this paper
• Dengue proteins with their role in pathogenesis, and strategies for developing an effective anti‐dengue treatment: A review

  2019influential citation
• Genetic and biochemical characterizations of Zika virus NS2A protein

  2019cites this paper
• Escape Mutations in NS 4 B Render Dengue Virus Insensitive to the Antiviral Activity of the Paracetamol Metabolite AM 404

  2019cites this paper
• Highly conserved epitopes of DENV structural and non-structural proteins: Candidates for universal vaccine targets

  2019cites this paper
• Mosquito cells persistently infected with dengue virus produce viral particles with host-dependent replication.

  2019cites this paper
• The Conserved Molecular Determinants of Virulence in Dengue Virus

  2019cites this paper
• The ER membrane protein complex is important for the biogenesis of flavivirus 1 polyproteins 2 3

  2019cites this paper
• The ER membrane protein complex is required to ensure correct topology and stable expression of flavivirus polyproteins

  2019cites this paper
• THE ACTIVITY OF AURORA KINASE B IS REQUIRED FOR DENGUE VIRUS RELEASE.

  2019cites this paper
• Recent update on anti-dengue drug discovery.

  2019cites this paper
• An insight into dengue virus proteins as potential drug/vaccine targets

  2019cites this paper
• A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication

  2019influential citation