Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Suppression of µ1 subunit of the adaptor protein complex 2 reduces dengue virus release.

Dengue virus (DENV) requires clathrin-mediated endocytosis for its entry into the cells where the adaptor protein complex (AP) is vital for the clathrin-coated vesicle formation. The role of AP-2 was previously examined in the early stages of DENV infection; however, the role of AP-2 in the late stage of DENV infection was not determined. The µ1 subunit of AP-2 (AP2M1) is one of the most important cytoplasmic carrier domains in clathrin-mediated endocytosis and the phosphorylation of this subunit by the kinase enzyme, AP-2 associated protein kinase 1 (AAK1), stimulates clathrin and supports the cell surface receptor incorporation. In the present study, we primarily aimed to investigate the role of AP2M1 by gene silencing approach as well as using naked DENV RNA transfection into AP2M1 knockdown cells. Secondarily, an inhibitor of AAK1, sunitinib was used to investigate whether AAK1 could influence the virus production in DENV-infected Huh7 cells. The knockdown of AP2M1 in the DENV-infected Huh7 cells displayed a reduction in the viral titer at 24 h post-infection. Furthermore, experiments were conducted to bypass the DENV internalization using a naked DENV RNA transfection into the AP2M1 knockdown cells. Higher intracellular DENV RNA, DENV E protein, and intracellular virion were observed, whereas the extracellular virion production was comparably less than that of control. Treatment with sunitinib in DENV-infected Huh7 cells was able to reduce extracellular virion production and was consistent with all four serotypes of DENV. Therefore, our findings demonstrate the role of AP2M1 in the exocytosis step of DENV replication leading to infectious DENV production and the efficacy of sunitinib in suppressing virus production during the infection with different serotypes of DENV.

Human glucose-regulated protein 78 modulates intracellular production and secretion of nonstructural protein 1 of dengue virus.

Virus-host interactions play important roles in virus infection and host cellular response. Several viruses, including dengue virus (DENV), usurp host chaperones to support their amplification and survival in the host cell. We investigated the interaction of nonstructural protein 1 (NS1) of DENV with three endoplasmic reticulum-resident chaperones (i.e. GRP78, calnexin and calreticulin) to delineate their functional roles and potential binding sites for protein complex formation. GRP78 protein showed prominent association with DENV NS1 in virus-infected Huh7 cells as evidenced by co-localization and co-immunoprecipitation assays. Further studies on the functional interaction of GRP78 protein were performed by using siRNA-mediated gene knockdown in a DENV replicon transfection system. GRP78 knockdown significantly decreased intracellular NS1 production and delayed NS1 secretion but had no effect on viral RNA replication. Dissecting the important domain of GRP78 required for DENV NS1 interaction showed co-immunoprecipitation of DENV NS1 with a full-length and substrate-binding domain (SBD), but not an ATPase domain, of GRP78, confirming their interaction through SBD binding. Molecular dynamics simulations of DENV NS1 and human GRP78 complex revealed their potential binding sites through hydrogen and hydrophobic bonding. The majority of GRP78-binding sites were located in a ß-roll domain and connector subdomains on the DENV NS1 structure involved in hydrophobic surface formation. Taken together, our findings demonstrated the roles of human GRP78 in facilitating the intracellular production and secretion of DENV NS1 as well as predicted potential binding sites between the DENV NS1 and GRP78 complex, which could have implications in the future development of target-based antiviral drugs.

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.

Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin-Mediated Neutralization.

Flavivirus nonstructural protein 1 (NS1) is a unique secreted nonstructural glycoprotein. Although it is absent from the flavivirus virion, intracellular and extracellular forms of NS1 have essential roles in viral replication and the pathogenesis of infection. The fate of NS1 in insect cells has been more controversial, with some reports suggesting it is exclusively cell associated. In this study, we confirm NS1 secretion from cells of insect origin and characterize its physical, biochemical, and functional properties in the context of dengue virus (DENV) infection. Unlike mammalian cell-derived NS1, which displays both high mannose and complex type N-linked glycans, soluble NS1 secreted from DENV-infected insect cells contains only high mannose glycans. Insect cell-derived secreted NS1 also has different physical properties, including smaller and more heterogeneous sizes and the formation of less stable NS1 hexamers. Both mammalian and insect cell-derived NS1 bind to complement proteins C1s, C4, and C4-binding protein, as well as to a novel partner, mannose-binding lectin. Binding of NS1 to MBL protects DENV against mannose-binding lectin-mediated neutralization by the lectin pathway of complement activation. As we detected secreted NS1 and DENV together in the saliva of infected Aedes aegypti mosquitoes, these findings suggest a mechanism of viral immune evasion at the very earliest phase of 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.

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.

Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication.

BACKGROUND: Host and viral proteins are involved in dengue virus (DENV) replication. Heterogeneous ribonucleoprotein (hnRNP) C1/C2 are abundant host cellular proteins that exhibit RNA binding activity and play important roles in the replication of positive-strand RNA viruses such as poliovirus and hepatitis C virus. hnRNP C1/C2 have previously been shown to interact with vimentin and viral NS1 in DENV-infected cells; however, their functional role in DENV replication is not clearly understood. In the present study, we investigated the role of hnRNP C1/C2 in DENV replication by using an in vitro model of DENV infection in a hepatocyte cell line (Huh7) and siRNA-mediated knockdown of hnRNP C1/C2. METHODS: Huh7 cells were transfected with hnRNP C1/C2-specific siRNA or irrelevant siRNA (control) followed by infection with DENV. Mock and DENV-infected knockdown cells were processed for immunoprecipitation using hnRNP C1/C2-specific antibody or their isotype-matched control antibody. The immunoprecipitated samples were subjected to RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR) for detection of DENV RNA. In addition, the knockdown cells harvested at varying time points after the infection were assessed for cell viability, cell proliferation, percentage of DENV infection, amount of viral RNA, and viral E and NS1 expression. Culture supernatants were subjected to focus forming unit assays to determine titers of infectious DENV. DENV luciferase reporter assay was also set up to determine viral translation. RESULTS: Immunoprecipitation with the anti-hnRNP C1/C2 antibody and subsequent RT-PCR revealed the presence of DENV RNA in the immunoprecipitated complex containing hnRNP C1/C2 proteins. Transfection with hnRNP C1/C2-specific siRNA resulted in a significant reduction of hnRNP C1/C2 mRNA and protein levels but did not induce cell death during DENV infection. The reduced hnRNP C1/C2 expression decreased the percentage of DENV antigen-positive cells as well as the amount of DENV RNA and the relative levels of DENV E and NS1 proteins; however, it had no direct effect on DENV translation. In addition, a significant reduction of DENV titers was observed in the supernatant from DENV-infected cells following the knockdown of hnRNP C1/C2. CONCLUSIONS: Our findings suggest that hnRNP C1/C2 is involved in DENV replication at the stage of viral RNA synthesis.

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.

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.

Dengue virus-induced hemorrhage in a nonhuman primate model.

Lack of a dengue hemorrhagic animal model recapitulating human dengue virus infection has been a significant impediment in advancing our understanding of the early events involved in the pathogenesis of dengue disease. In efforts to address this issue, a group of rhesus macaques were intravenously infected with dengue virus serotype 2 (strain 16 681) at 1 x 10(7) PFU/animal. A classic dengue hemorrhage developed 3 to 5 days after infection in 6 of 6 animals. Blood chemistry appeared to be normal with exception of creatine phosphokinase, which peaked at 7 days after infection. A modest thrombocytopenia and noticeable neutropenia concomitant with slight decrease of hemoglobin and hematocrit were registered. In addition, the concentration of D-dimer was elevated significantly. Viremia peaked at 3 to 5 days after infection followed by an inverse relationship between T and B lymphocytes and a bimodal pattern for platelet-monocytes and platelet-neutrophil aggregates. Dengue virus containing platelets engulfed by monocytes was noted at 8 or 9 days after infection. Thus, rhesus macaques inoculated intravenously with a high dose of dengue virus produced dengue hemorrhage, which may provide a unique platform to define the early events in dengue virus infection and help identify which blood components contribute to the pathogenesis of dengue disease.

Role of microparticles in dengue virus infection and its impact on medical intervention strategies.

Dengue virus (DV) is one of the most important vector-borne diseases in the world. It causes a disease that manifests as a spectrum of clinical symptoms, including dengue hemorrhagic fever. DV is proficient at diverting the immune system to facilitate transmission through its vector host, Aedes spp. mosquito. Similar to other vector-borne parasites, dengue may also require a second structural form, a virus of alternative morphology (VAM), to complete its life cycle. DV can replicate to high copy numbers in patient plasma, but no classical viral particles can be detected by ultra-structural microscopy analysis. A VAM appearing as a microparticle has been recapitulated with in vitro cell lines Meg01 and K562, close relatives to the cells harboring dengue virus in vivo. VAMs are likely to contribute to the high viremia levels observed in dengue patients. This review discusses the possible existence of a VAM in the DV life cycle.

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.

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.

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.

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 µ1A (AP-1 mu1A).

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO3⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO3⁻ exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease--distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 µ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.