Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells

デング2型ウイルスプロテアーゼを標的とした新規抗ウイルス薬探索のための蛍光発色によるアッセイ法の開発

Background: Dengue disease is one of the most significant vector-borne illnesses in the world. The emergence and re-emergence of dengue infections in many parts of the world affect millions annually and continue to burden public health systems especially in low-income populations. Advances in dengue vaccine development showed promising results; however, protection seems to be suboptimal. There is no licensed chemotherapeutic agent against dengue to date. An ideal scenario of combinatorial vaccination of high-risk individuals and chemotherapy of the diseased during outbreaks may compensate for the meager protection offered by the vaccine. The denguevirus protease is important to viral replication and, as such, has been identified as a potential target for antivirals. It is, therefore, our objective to establish and optimize an appropriate screening method for use during the earlystages of drug development for dengue. Methods: In this study, we developed and optimized a biochemical assay system for use in screening compound libraries against dengue virus protease. We tested the selected protease inhibitors with a cell-based assay to determine inhibition of viral replication. Results: We have presented direct plots of substrate kinetics data showing an apparent inhibition of the protease at excessive substrate concentrations. The most common sources of interference that may have affected the saidobservation were elucidated. Finally, a screen was done on an existing compound library using the developed method. The compounds selected in this study showed inhibitory activity against both the recombinant dengue protease and cell-based infectivity assays. Conclusions: Our study shows the practicality of a customized biochemical assay to find possible inhibitors of dengue viral protease during the initial stages of drug discovery.長崎大学学位論文 学位記番号:博(医歯薬)甲第890号 学位授与年月日:平成28年9月20日Author: Gianne Eduard L. Ulanday, Kenta Okamoto and Kouichi MoritaCitation: Tropical Medicine and Health, 44, 22; 2016Nagasaki University (長崎大学)課程博

The ZIIR Element of the Epstein-Barr Virus BZLF1 Promoter Plays a Central Role in Establishment and Maintenance of Viral Latency▿

The Epstein-Barr virus (EBV) BZLF1 gene encodes the immediate-early (IE) protein Zta, which plays a central role in regulating the switch between viral latency and lytic replication. A silencing element, ZIIR, is located between the ZID and ZII positive regulatory elements in the BZLF1 promoter Zp. We report here the phenotypes of variants of EBV strain B95.8 containing base substitution mutations in this ZIIR element. HEK293 cells infected with ZIIR mutant (ZIIRmt) virus produced at least 20-fold more viral IE Zta and Rta and early (E) EAD protein than did cells infected with the parental wild-type (WT) virus, leading to viral DNA replication and production of infectious virus. However, ZIIR mutant virus was 1/10 as efficient as WT virus in establishing proliferating B-cell clones following infection of human primary blood B cells. The ZIIRmt-infected lymphoblastoid cell lines (LCLs) that did grow out exhibited a phenotype similar to the one observed in 293 cells, including marked overproduction of IE and E gene products relative to WT-infected LCLs and lytic replication of the viral genome. Incubation of the ZIIRmt-infected LCLs with the chemical inducer 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to much greater activation of Zp than did the same treatment of WT- or ZVmt-infected LCLs. Furthermore, a protein kinase C (PKC) inhibitor, bis-indolylmaleimide, eliminated this activation by TPA. Thus, we conclude that ZIIR is a potent silencing element of Zp; it plays a key role in establishment and maintenance of EBV latency by inhibiting activation of Zp through the PKC signal transduction pathway