Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection.

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.

Melatonin Inhibits Dengue Virus Infection via the Sirtuin 1-Mediated Interferon Pathway.

Dengue virus (DENV) is the causative pathogen in the life-threatening dengue hemorrhagic fever and dengue shock syndrome. DENV is transmitted to humans via the bite of an infected Aedes mosquito. Approximately 100 million people are infected annually worldwide, and most of those live in tropical and subtropical areas. There is still no effective drug or vaccine for treatment of DENV infection. In this study, we set forth to investigate the effect of melatonin, which is a natural hormone with multiple pharmacological functions, against DENV infection. Treatment with subtoxic doses of melatonin dose-dependently inhibited DENV production. Cross-protection across serotypes and various cell types was also observed. Time-of-addition assay suggested that melatonin exerts its influence during the post-entry step of viral infection. The antiviral activity of melatonin partly originates from activation of the sirtuin pathway since co-treatment with melatonin and the sirtuin 1 (SIRT1) inhibitor reversed the effect of melatonin treatment alone. Moreover, melatonin could modulate the transcription of antiviral genes that aid in suppression of DENV production. This antiviral mechanism of melatonin suggests a possible new strategy for treating DENV infection.

Peptides targeting dengue viral nonstructural protein 1 inhibit dengue virus production.

Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries. Development of antiviral peptides against dengue virus (DENV) infection has previously been concentrated on blocking the actions of viral structural proteins and enzymes in virus entry and viral RNA processing in host cells. In this study, we proposed DENV NS1, which is a multifunctional non-structural protein indispensable for virus production, as a new target for inhibition of DENV infection by specific peptides. We performed biopanning assays using a phage-displayed peptide library and identified 11 different sequences of 12-mer peptides binding to DENV NS1. In silico analyses of peptide-protein interactions revealed 4 peptides most likely to bind to DENV NS1 at specific positions and their association was analysed by surface plasmon resonance. Treatment of Huh7 cells with these 4 peptides conjugated with N-terminal fluorescent tag and C-terminal cell penetrating tag at varying time-of-addition post-DENV infection could inhibit the production of DENV-2 in a time- and dose-dependent manner. The inhibitory effects of the peptides were also observed in other virus serotypes (DENV-1 and DENV-4), but not in DENV-3. These findings indicate the potential application of peptides targeting DENV NS1 as antiviral agents against DENV infection.

Serine protease inhibitor AEBSF reduces dengue virus infection via decreased cholesterol synthesis.

Dengue virus (DENV) infection has evolved into a major global health menace and economic burden due to its intensity and geographic distribution. DENV infection in humans can cause a wide range of symptoms including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An antiviral agent that is effective against all four serotypes of DENV is urgently needed to prevent and to manage this condition. Reducing the viral load during the early phase of infection may minimize the chance of patients progressing to more severe DHF or DSS. In this study, we set forth to investigate the anti-viral effect of five commercially available protease inhibitors on DENV infection since both viral and host proteases can contribute to effective viral replication. Previously, the serine protease inhibitor AEBSF [4-(2-aminoethyl) benzene sulfonyl fluoride] has been shown to inhibit DENV NS3 protease activity. The results of the present study revealed that DENV genome replication and protein synthesis were significantly inhibited by AEBSF in a dose-dependent manner. AEBSF inhibited the expression of genes such as 3-hydroxy 3-methyl-glutaryl-CoA synthase (HMGCS), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and low-density lipoprotein receptor (LDLR). Moreover, AEBSF significantly inhibited HMGCR activity and intracellular cholesterol synthesis after DENV infection. The anti-DENV effect of AEBSF was confirmed in all four DENV serotypes and in three different cell lines. These results indicate that AEBSF reduces DENV infection via both viral and host protease activities.

Vivo-morpholino oligomers strongly inhibit dengue virus replication and production.

Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3' stem-loop (3' SL) in the 3' UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 104-fold when compared to that of an untreated control. In addition, vivo-MOs also inhibited DENV production in U937 cells and primary human monocytes. Therefore, vivo-MOs targeting to the 3' SL in the 3' UTR of DENV genomes are effective and have the potential to be developed as anti-DENV agents.

RNAi screen reveals a role of SPHK2 in dengue virus-mediated apoptosis in hepatic cell lines.

Hepatic dysfunction is a feature of dengue virus (DENV) infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12), and sphingosine kinase 2 (SPHK2), were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

JNK1/2 inhibitor reduces dengue virus-induced liver injury.

High viral load with liver injury is exhibited in severe dengue virus (DENV) infection. Mitogen activated protein kinases (MAPKs) including ERK1/2 and p38 MAPK were previously found to be involved in the animal models of DENV-induced liver injury. However, the role of JNK1/2 signaling in DENV-induced liver injury has never been investigated. JNK1/2 inhibitor, SP600125, was used to investigate the role of JNK1/2 signaling in the BALB/c mouse model of DENV-induced liver injury. SP600125-treated DENV-infected mice ameliorated leucopenia, thrombocytopenia, hemoconcentration, liver transaminases and liver histopathology. DENV-induced liver injury exhibited induced phosphorylation of JNK1/2, whereas SP600125 reduced this phosphorylation. An apoptotic real-time PCR array profiler was used to screen how SP600125 affects the expression of 84 cell death-associated genes to minimize DENV-induced liver injury. Modulation of caspase-3, caspase-8 and caspase-9 expressions by SP600125 in DENV-infected mice suggests its efficiency in restricting apoptosis via both extrinsic and intrinsic pathways. Reduced expressions of TNF-α and TRAIL are suggestive to modulate the extrinsic apoptotic signals, where reduced p53 phosphorylation and induced anti-apoptotic Bcl-2 expression indicate the involvement of the intrinsic apoptotic pathway. This study thus demonstrates the pivotal role of JNK1/2 signaling in DENV-induced liver injury and how SP600125 modulates this pathogenesis.

Tyrosine kinase/phosphatase inhibitors decrease dengue virus production in HepG2 cells.

Dengue virus is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. High rates of dengue virus replication and virion production are related to disease severity. To identify anti-DENV compounds, we performed cell-based ELISA testing to detect the level of DENV E protein expression. Among a total of 83 inhibitors, eight were identified as inhibitors with antiviral activity. Epidermal growth factor receptor inhibitor II (EGFR/ErbB-2/ErbB-4 inhibitor II) and protein tyrosine phosphatase inhibitor IV (PTP inhibitor IV) significantly inhibited dengue virus production and demonstrated low toxicity in hepatocyte cell lines. Our results suggest the efficacy of tyrosine kinase/phosphatase inhibitors in decreasing dengue virus production in HepG2 cells.

Drug repurposing of minocycline against dengue virus infection.

Dengue virus infection is one of the most common arthropod-borne viral diseases. A complex interplay between host and viral factors contributes to the severity of infection. The antiviral effects of three antibiotics, lomefloxacin, netilmicin, and minocycline, were examined in this study, and minocycline was found to be a promising drug. This antiviral effect was confirmed in all four serotypes of the virus. The effects of minocycline at various stages of the viral life cycle, such as during viral RNA synthesis, intracellular envelope protein expression, and the production of infectious virions, were examined and found to be significantly reduced by minocycline treatment. Minocycline also modulated host factors, including the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2). The transcription of antiviral genes, including 2'-5'-oligoadenylate synthetase 1 (OAS1), 2'-5'-oligoadenylate synthetase 3 (OAS3), and interferon α (IFNA), was upregulated by minocycline treatment. Therefore, the antiviral activity of minocycline may have a potential clinical use against Dengue virus infection.

Mass spectrometric analysis of host cell proteins interacting with dengue virus nonstructural protein 1 in dengue virus-infected HepG2 cells.

Dengue virus (DENV) infection is a leading cause of the mosquito-borne infectious diseases that affect humans worldwide. Virus-host interactions appear to play significant roles in DENV replication and the pathogenesis of DENV infection. Nonstructural protein 1 (NS1) of DENV is likely involved in these processes; however, its associations with host cell proteins in DENV infection remain unclear. In this study, we used a combination of techniques (immunoprecipitation, in-solution trypsin digestion, and LC-MS/MS) to identify the host cell proteins that interact with cell-associated NS1 in an in vitro model of DENV infection in the human hepatocyte HepG2 cell line. Thirty-six novel host cell proteins were identified as potential DENV NS1-interacting partners. A large number of these proteins had characteristic binding or catalytic activities, and were involved in cellular metabolism. Coimmunoprecipitation and colocalization assays confirmed the interactions of DENV NS1 and human NIMA-related kinase 2 (NEK2), thousand and one amino acid protein kinase 1 (TAO1), and component of oligomeric Golgi complex 1 (COG1) proteins in virus-infected cells. This study reports a novel set of DENV NS1-interacting host cell proteins in the HepG2 cell line and proposes possible roles for human NEK2, TAO1, and COG1 in DENV infection.

SB203580 Modulates p38 MAPK Signaling and Dengue Virus-Induced Liver Injury by Reducing MAPKAPK2, HSP27, and ATF2 Phosphorylation.

Dengue virus (DENV) infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs), including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury.

Role of cathepsin B in dengue virus-mediated apoptosis.

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.

Compound A, a dissociated glucocorticoid receptor modulator, reduces dengue virus-induced cytokine secretion and dengue virus production.

Dengue Virus (DENV) infection is an important mosquito-borne viral disease and its clinical symptoms range from a predominantly febrile disease, dengue fever (DF), to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased levels of cytokines - the so-called 'cytokine storm', contribute to the pathogenesis of DHF/DSS. In this study, we compared the expression of cytokine genes between mock-infected and DENV-infected HepG2 cells using a real-time PCR array and revealed several up-regulated chemokines and cytokines, including CXCL10 and TNF-α. Compound A (CpdA), a plant-derived phenyl aziridine precursor containing anti-inflammatory action and acting as a dissociated nonsteroidal glucocorticoid receptor modulator, was selected as a candidate agent to modulate secretion of DENV-induced cytokines. CpdA is not a glucocorticoid but has an anti-inflammatory effect with no metabolic side effects as steroidal ligands. CpdA significantly reduced DENV-induced CXCL10 and TNF-α secretion and decreased leukocyte migration indicating for the first time the therapeutic potential of CpdA in decreasing massive immune activation during DENV infection.

Inhibition of p38MAPK and CD137 signaling reduce dengue virus-induced TNF-α secretion and apoptosis.

BACKGROUND: Hepatic injury in dengue virus (DENV) infection is authenticated by hepatomegaly and an upsurge in transaminase levels. DENV replicates in hepatocytes and causes hepatocyte apoptosis both in vitro and in vivo. Understanding the molecular mechanisms of DENV-induced hepatic injury could facilitate the development of alternate chemotherapeutic agents and improved therapies. FINDINGS: The p38 mitogen-activated protein kinase (MAPK) participates in both apoptosis-related signaling and pro- inflammatory cytokine production. The role of p38 MAPK in DENV-infected HepG2 cells was examined using RNA interference. The results showed that DENV infection activated p38 MAPK and induced apoptosis. The p38 MAPK activation and TNF-α production were controlled by p38 MAPK and CD137 signaling in DENV-infected HepG2 cells as activated p38 MAPK, TNF-α and apoptosis were significantly decreased in p38 MAPK and CD137 depleted DENV-infected HepG2 cells. Addition of exogenous TNF-α to p38 MAPK depleted DENV-infected HepG2 cells restored DENV-induced apoptosis in HepG2 cells. CONCLUSION: DENV induces CD137 signaling to enhance apoptosis by increasing TNF-α production via activation of p38 MAPK.

Multiploid CD61+ cells are the pre-dominant cell lineage infected during acute dengue virus infection in bone marrow.

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

Infection of bone marrow cells by dengue virus in vivo.

Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41(+)CD61(+) cells, regardless of CD34 and CD45 expression. The CD61(+) cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting-assisted isolation of CD61(+) cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection.

Role of CD137 signaling in dengue virus-mediated apoptosis.

Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

Cell death gene expression profile: role of RIPK2 in dengue virus-mediated apoptosis.

Dengue virus (DENV) is a major emerging arthropod-borne pathogen, which infects individuals in both subtropical and tropical regions. Patients with DENV infection exhibit evidence of hepatocyte injury. However, the mechanisms of hepatocyte injury are unclear. Therefore we examined the expression of cell death genes during DENV-infection of HepG2 cells using real-time PCR arrays. The expression changes were consistent with activation of apoptosis and autophagy. Expression of the up-regulated genes, including RIPK2, HRK, TGF-ß, PERK, and LC3B, was confirmed by quantitative real-time PCR. RIPK2 belongs to the receptor-interacting protein family of serine/threonine protein kinases, which is a crucial mediator of multiple stress responses that leads to the activation of caspase, NF-κB and MAP kinases including JNK and p38. RIPK2 activity is inhibited by the p38 MAPK pathway inhibitor SB203580. The effect of SB203580 on RIPK2 expression and DENV-induced apoptosis was tested in DENV-infected HepG2 cells. The inhibition of RIPK2 expression by SB203580 significantly reduced apoptosis. SB203580 also significantly reduced DENV capsid protein (DENVC)-mediated apoptosis. Suppression of endogenous RIPK2 in DENV-infected HepG2 cells by small interfering RNA (siRNA) significantly decreased apoptosis suggesting for the first time that RIPK2 plays a role in DENV-mediated apoptosis.

Interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production.

Dengue virus infection is an important mosquito-borne disease and a public health problem worldwide. A better understanding of interactions between human cellular host and dengue virus proteins will provide insight into dengue virus replication and cellular pathogenesis. The glycosylated envelope protein of dengue virus, DENV E, is processed in the endoplasmic reticulum of host cells and therefore reliant on host processing functions. The complement of host ER functions involved and nature of the interactions with DENV E has not been thoroughly investigated. By employing a yeast two-hybrid assay, we found that domain III of DENV E interacts with human immunoglobulin heavy chain binding protein (BiP). The relevance of this interaction was demonstrated by co-immunoprecipitation and co-localization of BiP and DENV E in dengue virus-infected cells. Using the same approach, association of DENV E with two other chaperones, calnexin and calreticulin was also observed. Knocking-down expression of BiP, calnexin, or calreticulin by siRNA significantly decreased the production of infectious dengue virions. These results indicate that the interaction of these three chaperones with DENV E plays an important role in virion production, likely facilitating proper folding and assembly of dengue proteins.

Association of dengue virus NS1 protein with lipid rafts.

During the replication of dengue virus, a viral non-structural glycoprotein, NS1, associates with the membrane on the cell surface and in the RNA replication complex. NS1 lacks a transmembrane domain, and the mechanism by which it associates with the membrane remains unclear. This study aimed to investigate whether membrane-bound NS1 is present in lipid rafts in dengue virus-infected cells. Double immunofluorescence staining of infected HEK-293T cells revealed that NS1 localized with raft-associated molecules, ganglioside GM1 and CD55, on the cell surface. In a flotation gradient centrifugation assay, a small proportion of NS1 in Triton X-100 cell lysate consistently co-fractionated with raft markers. Association of NS1 with lipid rafts was detected for all four dengue serotypes, as well as for Japanese encephalitis virus. Analysis of recombinant NS1 forms showed that glycosylated NS1 dimers stably expressed in HEK-293T cells without an additional C-terminal sequence, or with a heterologous transmembrane domain, failed to associate with lipid rafts. In contrast, glycosylphosphatidylinositol-linked recombinant NS1 exhibited a predilection for lipid rafts. These results indicate an association of a minor subpopulation of NS1 with lipid rafts during dengue virus infection and suggest that modification of NS1, possibly lipidation, is required for raft association.