Ching-Len Liao

Japanese Encephalitis Virus Infection Initiates Endoplasmic Reticulum Stress and an Unfolded Protein Response

ABSTRACT The malfunctioning of the endoplasmic reticulum (ER) of cells in hosts ranging from yeast to mammals can trigger an unfolded protein response (UPR). Such malfunctioning can result from a variety of ER stresses, including the inhibition of protein glycosylation and calcium imbalance. To cope with ER stresses, cells may rely on the UPR to send a signal(s) from the ER to the nucleus to stimulate appropriate cellular responses, including induction of chaperone expression. During Japanese encephalitis virus (JEV) infection, the lumen of the ER rapidly accumulates substantial amounts of viral proteins for virus progeny production. In the present study, we demonstrate that as evidenced by certain chaperone inductions, JEV infection triggers the UPR in fibroblast BHK-21 cells and in neuronal N18 and NT-2 cells, in which JEV results in apoptotic cell death. By contrast, no UPR was observed in apoptosis-resistant K562 cells infected by JEV. JEV infection also activates expression of CHOP/GADD153, a distinctive transcription factor often induced by the UPR, and appears to trigger activation of p38 mitogen-activated protein kinase, a posttranslational activator of CHOP. Ectopic enforcement of CHOP expression enhanced JEV-induced apoptosis, whereas treatment with a p38-specific inhibitor, SB203580, partially blocked JEV-induced apoptosis. Interestingly, bcl-2 overexpression and treatment with a pancaspase inhibitor, z-VAD-fmk, inhibited CHOP induction and diminished JEV-induced apoptosis, suggesting that Bcl-2 and caspases could be the upstream regulators of CHOP. Our results thus suggest that virus-induced ER stress may participate, via p38-dependent and CHOP-mediated pathways, in the apoptotic process triggered by JEV infection.

Blocking of Interferon-Induced Jak-Stat Signaling by Japanese Encephalitis Virus NS5 through a Protein Tyrosine Phosphatase-Mediated Mechanism

ABSTRACT Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes severe human disease, has been shown to block the interferon (IFN)-induced Janus kinase signal transducer and activation of transcription (Jak-Stat) signaling cascade by preventing Tyk2 tyrosine phosphorylation and Stat activation. In this study, we demonstrate that expression of the JEV nonstructural protein NS5 readily blocked IFN-stimulated Jak-Stat signaling events such as Stat1 nuclear translocation and tyrosine phosphorylation of Tyk2 and Stat1. The region of JEV NS5 responsible for Stat1 suppression was identified using various deletion clones. Deletion of 83 N-terminal residues of JEV NS5, but not the 143 C-terminal residues, abolished its ability to block IFN-stimulated Stat1 activation. The role of JEV NS5 as an IFN antagonist was further demonstrated by its ability to block the induction of interferon-stimulated genes and the antiviral effect of IFN-α against the IFN-sensitive encephalomyocarditis virus, which appears to replicate and kill cells that express NS5 even with alpha IFN treatment. Furthermore, the molecular mechanism responsible for IFN antagonism by NS5 probably involves protein tyrosine phosphatases (PTPs), as the IFN-blocking events in both JEV-infected and NS5-expressing cells were reversed by sodium orthovanadate, a broad-spectrum inhibitor of PTPs. We suggest that JEV NS5 is an IFN antagonist and that it may play a role in blocking IFN-stimulated Jak-Stat signaling via activation of PTPs during JEV infection.

Antiviral Effects of an Iminosugar Derivative on Flavivirus Infections

ABSTRACT Endoplasmic reticulum (ER) α-glucosidase inhibitors, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of several ER-budding viruses. Here we investigated the effects of one such inhibitor, N-nonyl-deoxynojirimycin (NN-DNJ), a 9-carbon alkyl iminosugar derivative, on infection by Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2). In the presence of NN-DNJ, JEV and DEN-2 infections were suppressed in a dose-dependent manner. This inhibitory effect appeared to influence DEN-2 infection more than JEV infection, since lower concentrations of NN-DNJ substantially blocked DEN-2 replication. Secretion of the flaviviral glycoproteins E and NS1 was greatly reduced, and levels of DEN-2 viral RNA replication measured by fluorogenic reverse transcription-PCR were also decreased, by NN-DNJ. Notably, the viral glycoproteins, prM, E, and NS1 were found to associate transiently with the ER chaperone calnexin, and this interaction was affected by NN-DNJ, suggesting a potential role of calnexin in the folding of flaviviral glycoproteins. Additionally, in a mouse model of lethal challenge by JEV infection, oral delivery of NN-DNJ reduced the mortality rate. These findings show that NN-DNJ has an antiviral effect on flavivirus infection, likely through interference with virus replication at the posttranslational modification level, occurring mainly in the ER.

Blocking of the Alpha Interferon-Induced Jak-Stat Signaling Pathway by Japanese Encephalitis Virus Infection

ABSTRACT The induction of alpha/beta interferon (IFN-α/β) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-α had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-α in the cultured cell system. JEV infection appeared to render cells resistant to IFN-α since the IFN-α-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-α/β receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-α stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-α/β by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway.

Cholesterol Effectively Blocks Entry of Flavivirus

ABSTRACT Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2) are enveloped flaviviruses that enter cells through receptor-mediated endocytosis and low pH-triggered membrane fusion and then replicate in intracellular membrane structures. Lipid rafts, cholesterol-enriched lipid-ordered membrane domains, are platforms for a variety of cellular functions. In this study, we found that disruption of lipid raft formation by cholesterol depletion with methyl-β-cyclodextrin or cholesterol chelation with filipin III reduces JEV and DEN-2 infection, mainly at the intracellular replication steps and, to a lesser extent, at viral entry. Using a membrane flotation assay, we found that several flaviviral nonstructural proteins are associated with detergent-resistant membrane structures, indicating that the replication complex of JEV and DEN-2 localizes to the membranes that possess the lipid raft property. Interestingly, we also found that addition of cholesterol readily blocks flaviviral infection, a result that contrasts with previous reports of other viruses, such as Sindbis virus, whose infectivity is enhanced by cholesterol. Cholesterol mainly affected the early step of the flavivirus life cycle, because the presence of cholesterol during viral adsorption greatly blocked JEV and DEN-2 infectivity. Flavirial entry, probably at fusion and RNA uncoating steps, was hindered by cholesterol. Our results thus suggest a stringent requirement for membrane components, especially with respect to the amount of cholesterol, in various steps of the flavivirus life cycle.

Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity

Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

Flavivirus Activates Phosphatidylinositol 3-Kinase Signaling To Block Caspase-Dependent Apoptotic Cell Death at the Early Stage of Virus Infection

ABSTRACT Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an antiapoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.

Distinct Antiviral Roles for Human 2′,5′-Oligoadenylate Synthetase Family Members against Dengue Virus Infection

The 2′,5′-oligoadenylate synthetase (OAS) and its downstream effector RNase L play important roles in host defense against virus infection. Oas1b, one of the eight Oas1 genes in the mouse genome, has been identified as a murine flavivirus-resistance gene. Four genes, OAS1, OAS2, OAS3, and OAS-like (OASL), have been identified in the human OAS gene family, and 10 isoforms, including OAS1 (p42, p44, p46, p48, and p52), OAS2 (p69 and p71), OAS3 (p100), and OASL (p30 and p59) can be generated by alternative splicing. In this study, we determined the role of the human OAS/RNase L pathway in host defense against dengue virus (DEN) infection and assessed the antiviral potential of each isoform in the human OAS family. DEN replication was reduced by overexpression and enhanced by knockdown of RNase L expression, indicating a protective role for RNase L against DEN replication in human cells. The human OAS1 p42, OAS1 p46, and OAS3 p100, but not the other OAS isoforms, blocked DEN replication via an RNase L-dependent mechanism. Furthermore, the anti-DEN activities of these three OAS isoforms correlated with their ability to trigger RNase L activation in DEN-infected cells. Thus, OAS1 p42/p46 and OAS3 p100 are likely to contribute to host defense against DEN infection and play a role in determining the outcomes of DEN disease severity.

Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice.

To evaluate the potential of DNA vaccine against dengue (DEN) infection, we characterize the protective efficacy and immune responses of mice intramuscularly injected with plasmid encoding DEN-2 non-structural protein 1 (NS1). Intravenously challenged by lethal DEN-2, mice vaccinated with NS1-DNA exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. In addition to a moderate increase of NS1-specific antibody titer from immunized mice measured by ELISA, a strong priming effect on anti-NS1 response was also noticed in plasmid NS1-vaccinated mice by radioimmunoprecipitation (RIP) or immunoblot analysis. Interestingly, newborn mice from NS1-DNA-immunized dam showed stronger resistance to viral challenge, as compared to those from vector DNA or PBS-immunized dams, indicating the protective role of NS1-specific antibody. In contrast to humoral immune response, DNA immunization can elicit strong cellular immune responses, including NS1-specific T cell proliferation and cytolytic activity. The NS1-DNA-induced protection can be further augmented by co-injection of plasmid encoding interleukin 12 (IL-12), suggesting an effector role of Th1 immunity against DEN infection. In summary, our results suggest the potential of NS1-DNA vaccine against DEN infection, and indicate both NS1-specific humoral and cellular immune responses contribute to the protection.

Novel Dengue Virus-Specific NS2B/NS3 Protease Inhibitor, BP2109, Discovered by a High-Throughput Screening Assay

ABSTRACT Dengue virus (DENV) causes disease globally, with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available, and treatment is supportive. In this study, we identified BP2109, a potent and selective small-molecule inhibitor of the DENV NS2B/NS3 protease, by a high-throughput screening assay using a recombinant protease complex consisting of the central hydrophilic portion of NS2B and the N terminus of the protease domain. BP2109 inhibited DENV (serotypes 1 to 4), but not Japanese encephalitis virus (JEV), replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with a 50% inhibitory concentration (IC50) of 15.43 ± 2.12 μM and reduced the reporter expression of the DENV-2 replicon with a 50% effective concentration (EC50) of 0.17 ± 0.01 μM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of the NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue virus NS2B/NS3 protease and a dengue virus replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined to be the key mutation conferring dengue virus replicon resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect resistance to BP2109. Both the R55K and E80K mutations are located in the central hydrophilic portion of the NS2B cofactor, where extensive interactions with the NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.