The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

The vitamin D receptor agonist EB1089 can exert its antiviral activity independently of the vitamin D receptor.

Vitamin D has been shown to have antiviral activity in a number of different systems. However, few studies have investigated whether the antiviral activity is exerted through the vitamin D receptor (VDR). In this study, we investigated whether the antiviral activity of a vitamin D receptor agonist (EB1089) towards dengue virus (DENV) was modulated by VDR. To undertake this, VDR was successively overexpressed, knocked down and retargeted through mutation of the nuclear localization signal. In no case was an effect seen on the level of the antiviral activity induced by EB1089, strongly indicating that the antiviral activity of EB1089 is not exerted through VDR. To further explore the antiviral activity of EB1089 in a more biologically relevant system, human neural progenitor cells were differentiated from induced pluripotent stem cells, and infected with Zika virus (ZIKV). EB1089 exerted a significant antiviral effect, reducing virus titers by some 2Log10. In support of the results seen with DENV, no expression of VDR at the protein level was observed. Collectively, these results show that the vitamin D receptor agonist EB1089 exerts its antiviral activity independently of VDR.

Investigation of the activity of baicalein towards Zika virus.

BACKGROUND: Zika virus (ZIKV) is a mosquito transmitted virus spread primarily by Aedes species mosquitoes that can cause disease in humans, particularly when infection occurs in pregnancy where the virus can have a significant impact on the developing fetus. Despite this, there remains no prophylactic agent or therapeutic treatment for infection. Baicalein is a trihydroxyflavone, that is found in some traditional medicines commonly used in Asia, and has been shown to have several activities including antiviral properties. Importantly, studies have shown baicalein to be safe and well tolerated in humans, increasing its potential utilization. METHODS: This study sought to determine the anti-ZIKV activity of baicalein using a human cell line (A549). Cytotoxicity of baicalein was determined by the MTT assay, and the effect on ZIKV infection determined by treating A549 cells with baicalien at different time points in the infection process. Parameters including level of infection, virus production, viral protein expression and genome copy number were assessed by flow cytometry, plaque assay, western blot and quantitative RT-PCR, respectively. RESULTS: The results showed that baicalein had a half-maximal cytotoxic concentration (CC50) of > 800 µM, and a half-maximal effective concentration (EC50) of 124.88 µM. Time-of-addition analysis showed that baicalein had an inhibitory effect on ZIKV infection at the adsorption and post-adsorption stages. Moreover, baicalein also exerted a significant viral inactivation activity on ZIKV (as well as on dengue virus and Japanese encephalitis virus) virions. CONCLUSION: Baicalein has now been shown to possess anti-ZIKV activity in a human cell line.

Activity of vitamin D receptor agonists against dengue virus.

Infections with the mosquito-transmitted dengue virus (DENV) are a pressing public health problem in many parts of the world. The recently released commercial vaccine for DENV has encountered some problems, and there is still no effective drug to treat infections. Vitamin D has a well characterized role in calcium and phosphorus homeostasis, but additionally has a role in the immune response to bacterial and viral pathogens. In this study a number of fused bicyclic derivatives of 1H-pyrrolo[1,2]imidazol-1-one with vitamin D receptor (VDR) agonist activity were evaluated for possible anti-DENV activity. The results showed that five of the compounds were able to significantly inhibit DENV infection. The most effective compound, ZD-3, had an EC50 value of 7.47 µM and a selective index of 52.75. The compounds were only effective when used as a post-infection treatment and treatment significantly reduced levels of infection, virus output, DENV protein expression and genome copy number. These results suggest that these VDR agonists have the potential for future development as effective anti-DENV agents.

Discordant Activity of Kaempferol Towards Dengue Virus and Japanese Encephalitis Virus.

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.

Analysis of Zika virus neutralizing antibodies in normal healthy Thais.

Zika virus (ZIKV) infections have been reported from all over Thailand, but the number of reported cases remains low, suggesting a degree of immune protection against ZIKV infection. To address this possibility, the presence of ZIKV neutralizing antibodies was determined in serum from 135 healthy Thai adults with a plaque reduction neutralization test (PRNT), and a number of samples were subsequently analyzed for the presence of neutralizing antibodies to dengue virus (DENV) and Japanese encephalitis virus (JEV). Results showed that 70.4% (PRNT50 ≥ 10), 55.6 (PRNT50 ≥ 20) or 22.2% (PRNT90 ≥ 20) of the samples showed neutralizing antibodies to ZIKV. Detailed analysis showed no association between the presence of neutralizing antibodies to other flaviviruses (DENV, JEV) and the presence of ZIKV neutralizing antibodies. These results suggest that the level of ZIKV neutralizing antibodies in the Thai population is enough to dampen the transmission of the virus in Thailand.

Polyketides and Alkaloids from the Marine-Derived Fungus Dichotomomyces cejpii F31-1 and the Antiviral Activity of Scequinadoline A against Dengue Virus.

In our continuous chemical investigation on the marine-derived fungus Dichotomomyces cejpii F31-1, two new polyketides dichocetides B-C (1, 2), two new alkaloids dichotomocejs E-F (3, 4), and three known fumiquinozalines: scequinadoline A (5), quinadoline A (6), and scequinadoline E (7) were discovered from the culture broth and the mycelium in the culture medium, by the addition of l-tryptophan and l-phenylalanine. Their chemical structures were established by one dimensional (1D), two dimensional (2D) nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS) data. Among them, scequinadoline A (5) exhibited significant inhibitory activity against dengue virus serotype 2 production by standard plaque assay, equivalent to the positive control andrographlide. Scequinadoline A (5) possesses the potential for further development as a dengue virus inhibitor.

Modulation of hepcidin expression by normal control and beta0-thalassemia/Hb E erythroblasts.

OBJECTIVES: The inherited genetic disorder beta0-thalassemia/Hb E disease is associated with the over-suppression of the master regulator of iron homeostasis, the peptide hormone hepcidin. How developing erythroid cells mediate the suppression of hepcidin remains controversial, although a number of inhibitors have been proposed. METHODS: To investigate the ability of erythroid cells to suppress hepcidin expression in liver cells, conditioned media from the culture of in vitro differentiating erythroblasts (from normal controls and beta0-thalassemia/Hb E patients) was used to treat HepG2 cells, and the effects on hepcidin expression were assayed by real-time quantitative PCR and confocal microscopy. RESULTS: Early activation followed by later suppression of hepcidin expression was seen posttreatment. Markedly, however, no significant differences were observed between suppression of hepcidin as mediated by media from the culture of erythroblasts from normal controls and beta0-thalassemia/Hb E patients Discussion: Previous studies investigating the suppression of hepcidin expression in beta0-thalassemia/Hb E disease have used patient-derived serum samples, which are complex fluids with contributions from multiple cell types. This study has developed a simple in vitro system that allows investigation of how a single cell type mediates hepcidin expression. The results support proposals that over-suppression of hepcidin seen in beta-thalassemia/Hb E patients is a consequence of the increased mass of erythropoiesis and not defects in the signaling process per se. CONCLUSION: The in vitro cell system developed here allows further investigation into the processes mediating erythroid cell suppression of liver hepcidin expression in both normal and pathological states.

Mitochondrial Changes in ß0-Thalassemia/Hb E Disease.

The compound ß°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and ß°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from ß°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from ß°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.

Dysregulation of ferroportin gene expression in ß(0)-thalassemia/Hb E disease.

During erythropoiesis, iron levels need to be carefully regulated to ensure there is sufficient iron available for hemoglobin synthesis, but that there is no excess to cause damage to the developing erythroblast. Iron influx to the developing erythroblast is controlled by the expression of the transferrin receptor, while iron efflux is regulated by ferroportin (FPN), the sole iron-exporting protein. FPN is encoded through multiple messenger RNAs (mRNAs) some of which contain an iron-responsive element (variant I mRNAs) and some of which do not (variant II mRNAs). This study sought to investigate the expression of the FPN mRNAs in developing erythroblasts from normal controls and ß(0)-thalassemia/Hb E patients. While levels of FPN protein were relatively constant, marked reductions of the variant I message were seen in erythroblasts from ß(0)-thalassemia/Hb E patients as compared to normal control cells, particularly in late erythropoiesis. Variant II mRNAs were generally increased during erythroid differentiation. No difference was seen in levels of either transferrin or ferritin heavy chain expression. While no difference was observed in labile iron pools under normal culture conditions, erythroblasts from ß(0)-thalassemia/Hb E patients showed a significantly reduced expression of total FPN message under high iron conditions as compared to normal control erythroblasts. These results are consisted with dysregulation of iron efflux from the maturing erythroblast in ß(0)-thalassemia/Hb E patients, and this dysregulation possibly contributes to ineffective erythropoiesis seen in these patients.