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

Chemistry Central Journal Poster presentation Molecular modeling studies on dengue and West Nile Virus NS2B/NS3 protease inhibitor interaction

Dengue Virus (DV) and West Nile Virus (WNV) are causes of severe public-health problems in many countries. Neither effective human vaccines nor drugs against both DV and WNV are available. NS2B (an essential cofactor) together with NS3 protease, which is a vital enzyme for the viruses in the replication cycle, is currently focused as the promising enzyme target for drug development. However, the active enzyme-inhibitor complex of DV is not yet experimentally available. Therefore, 3D structure of the DV's enzyme was built using the related WNV NS2B/NS3 structure as a template (2FP7.pdb) [1]. The DV model and 2FP7.pdb (WNV-X) were subjected to molecular dynamics (MD) simulations. MD simulations reveal the important of NS2B for interacting with both NS3 protease and P2 subsite of inhibitor. The final MD snapshot of D

The marriage of chemokines and galectins as functional heterodimers

Trafficking of leukocytes and their local activity profile are of pivotal importance for many (patho)physiological processes. Fittingly, microenvironments are complex by nature, with multiple mediators originating from diverse cell types and playing roles in an intimately regulated manner. To dissect aspects of this complexity, effectors are initially identified and structurally characterized, thus prompting familial classification and establishing foci of research activity. In this regard, chemokines present themselves as role models to illustrate the diversification and fine-tuning of inflammatory processes. This in turn discloses the interplay among chemokines, their cell receptors and cognate glycosaminoglycans, as well as their capacity to engage in new molecular interactions that form hetero-oligomers between themselves and other classes of effector molecules. The growing realization of versatility of adhesion/growth-regulatory galectins that bind to glycans and proteins and their presence at sites of inflammation led to testing the hypothesis that chemokines and galectins can interact with each other by protein-protein interactions. In this review, we present some background on chemokines and galectins, as well as experimental validation of this chemokine-galectin heterodimer concept exemplified with CXCL12 and galectin-3 as proof-of-principle, as well as sketch out some emerging perspectives in this arena

Histone H3 Cleavage in Severe COVID-19 ICU Patients

The severity of coronavirus disease 19 (COVID-19) is associated with neutrophil extracellular trap (NET) formation. During NET formation, cytotoxic extracellular histones are released, the presence of which is linked to the initiation and progression of several acute inflammatory diseases. Here we study the presence and evolution of extracellular histone H3 and several other neutrophil-related molecules and damage-associated molecular patterns (DAMPs) in the plasma of 117 COVID-19-positive ICU patients. We demonstrate that at ICU admission the levels of histone H3, MPO, and DNA-MPO complex were all significantly increased in COVID-19-positive patients compared to control samples. Furthermore, in a subset of 54 patients, the levels of each marker remained increased after 4+ days compared to admission. Histone H3 was found in 28% of the patients on admission to the ICU and in 50% of the patients during their stay at the ICU. Notably, in 47% of histone-positive patients, we observed proteolysis of histone in their plasma. The overall presence of histone H3 during ICU stay was associated with thromboembolic events and secondary infection, and non-cleaved histone H3 was associated with the need for vasoactive treatment, invasive ventilation, and the development of acute kidney injury. Our data support the validity of treatments that aim to reduce NET formation and additionally underscore that more targeted therapies focused on the neutralization of histones should be considered as treatment options for severe COVID-19 patients.</p