Dengue virus disrupts Daxx and NF-κB interaction to induce CD137-mediated apoptosis.

Dengue virus (DENV) is a positive-strand RNA virus of the Flavivirus family with 4 different serotypes. Clinical manifestations of DENV infection include dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Following DENV infection, apoptosis of hepatic cells is observed both in vitro and in vivo. However, the molecular mechanisms revealing how viral components affect cellular apoptosis remain unclear. In the present study, the role of death domain-associated protein 6 (Daxx) in DENV-mediated apoptosis was characterized by RNA interference and overexpression studies, and the anti-apoptotic function of Daxx during DENV infection was identified. Furthermore, the viral component, DENV capsid protein (DENV C), interacted with Daxx to disrupt interaction between Daxx and NF-κB. The liberated NF-κB activated the promoter of CD137, which is a member of the TNF family, and is previously shown to induce apoptosis during DENV infection. In summary, DENV C disrupts Daxx and NF-κB interaction to induce CD137-mediated apoptosis 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.

Adaptor protein 1 complexes regulate intracellular trafficking of the kidney anion exchanger 1 in epithelial cells.

Distal renal tubular acidosis (dRTA) can be caused by mutations in the gene encoding the anion exchanger 1 (AE1) and is characterized by defective urinary acidification, metabolic acidosis, and renal stones. AE1 is expressed at the basolateral membrane of type A intercalated cells in the renal cortical collecting duct (kAE1). Two dRTA mutations result in the carboxyl-terminal truncation of kAE1; in one case, the protein trafficked in a nonpolarized way in epithelial cells. A recent yeast two-hybrid assay showed that the carboxyl-terminal cytosolic domain of AE1 interacts with adaptor protein complex 1 (AP-1A) subunit µ1A (mu-1A; Sawasdee N, Junking M, Ngaojanlar P, Sukomon N, Ungsupravate D, Limjindaporn T, Akkarapatumwong V, Noisakran S, Yenchitsomanus PT. Biochem Biophys Res Commun 401: 85-91, 2010). Here, we show the interaction between kAE1 and mu-1A and B in vitro by reciprocal coimmunoprecipitation in epithelial cells and in vivo by coimmunoprecipitation from mouse kidney extract. When endogenous mu-1A (and to a lesser extent mu-1B) was reduced, kAE1 protein was unable to traffic to the plasma membrane and was rapidly degraded via a lysosomal pathway. Expression of either small interfering RNA-resistant mu-1A or mu-1B stabilized kAE1 in these cells. We also show that newly synthesized kAE1 does not traffic through recycling endosomes to the plasma membrane, suggesting that AP-1B, located in recycling endosomes, is not primarily involved in trafficking of newly synthesized kAE1 when AP-1A is present in the cells. Our data demonstrate that AP-1A regulates processing of the basolateral, polytopic membrane protein kAE1 to the cell surface and that both AP-1A and B adaptor complexes are required for normal kAE1 trafficking.

Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production.

Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.

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.

Nuclear localization of dengue virus capsid protein is required for DAXX interaction and apoptosis.

Dengue virus capsid protein (DENVC) localizes to both the cytoplasm and nucleus of dengue virus-infected cells. DENV C contains three nuclear localization signals (NLS), (6)KKAR(9), (73)KKSK(76), and the bipartite signal (85)RKeigrmlnilnRRRR(100). Stable HepG2 cells constitutively expressing DENV C, DENV C (Delta 85-100) and DENV C (Delta 73-100) were constructed to clarify whether nuclear translocation of DENV C affected apoptosis in liver cell line. While the wild-type DENV C could translocate into the nuclei of HepG2 cells, the mutant DENV Cs were restricted to the cytoplasm. The loss of nuclear localization of both mutant DENV Cs resulted in the disruption of their interactions with the apoptotic protein Daxx. Interestingly, upon treatment with anti-Fas antibody, the HepG2 cells expressing the wild-type DENV C showed significantly more apoptosis compared with the HepG2 cells expressing either mutant DENV C. To identify the amino acids required for DAXX interaction and apoptosis, substitution mutations either (K73A/K74A) or (R85A/K86A) were introduced into the C-terminal region of DENV C, and tested whether these mutations affected its interaction with Daxx and apoptosis. The results demonstrate that (73)KK and (85)RK of DENV C are important for its nuclear localization, interaction with DAXX and induction of apoptosis. This work is the first to demonstrate that nuclear localization of DENV C is required for DAXX interaction and apoptosis.

Production and characterization of anti-dengue capsid antibodies suggesting the N terminus region covering the first 20 amino acids of dengue virus capsid protein is predominantly immunogenic in mice.

We produced monoclonal and polyclonal antibodies to the capsid (C) protein of dengue serotype 2 virus (DV2 C). First, a maltose-binding protein fused to DV2 C protein (MBP-C) was overproduced in E. coli. The affinity-purified MBP-C protein was cleaved by factor Xa protease to obtain a recombinant DV2 C protein, which was then used for mouse immunizations. Two hybridoma cell lines producing anti-C Mabs as well as anti-C polyclonal antibody were successfully generated and characterized. Interestingly, all of the generated antibodies specifically recognized the first 20 amino acids of the DV2 C protein, as determined by peptide epitope mapping and via a recombinant DV2 C protein in which this region was deleted. The results suggested that this region is predominantly immunogenic in mice.

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.

Sensitization to Fas-mediated apoptosis by dengue virus capsid protein.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are important public health problems in tropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF. However, hepatomegaly, hepatocellular necrosis and fulminant hepatic failure are occasionally observed in patients with DHF. Dengue virus-infected liver cells undergo apoptosis but the underlying molecular mechanism remains unclear. Using a yeast two-hybrid screen, we found that dengue virus capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx, a Fas-associated protein. The interaction between DENV C and Daxx in dengue virus-infected liver cells was also demonstrated by co-immunoprecipitation and double immunofluorescence staining. The two proteins were predominantly co-localized in the cellular nuclei. Fas-mediated apoptotic activity in liver cells constitutively expressing DENV C was induced by anti-Fas antibody, indicating that the interaction of DENV C and Daxx involves in apoptosis of dengue virus-infected liver cells.

The distribution of IL-10 promoter polymorphism in Thais.

IL-10 is a regulatory cytokine, which plays important roles in the pathogenesis of many diseases polymorphism of IL-10 promoter influences the phenotypic expressions such as the variation of IL-10 production among individuals and is subjected to the genetic susceptibility study of many diseases. However, there is no information about the frequencies of IL-10 promoter polymorphism in a Thai population. To determine the distribution of IL-10 promoter polymorphism in unrelated healthy Thais, genomic-DNA from 160 unrelated healthy volunteers were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The functional single-nucleotide polymorphisms (SNPs) in IL-10 promoter (positions -1082 (G/A), -819 (C/T), -592 (C/A) were included. The allele frequencies of -1082*A (93.4%), -819*T (71.9%), and -592*A (71.9%) were significantly higher than the allele frequencies of -1082*G (6.6%), -819*C (28.1%), and -592*C (28.1%) respectively in a Thai population similar to other Asian populations (Korean, Japanese, Chinese). As for the haplotype analysis, the ATA haplotype (72%) was significantly higher in Thais and other Asian populations compared to non-Asian populations; whereas, GCC haplotype (6.6%) was significantly lower in Thais. Additionally, two rare haplotypes of IL-10 promoter (ATC and ACA) which were previously reported only in the Chinese Han people, were found with similar frequencies (0.6%) in the present study. In conclusion, the distribution of IL-10 promoter polymorphisms in Thais was comparable to other Asian populations but distinct from Non-Asian populations. At least five haplotypes existed in an unrelated healthy Thai population as ACC, GCC, ATA, ATC, and ACA haplotypes.