Genetic and functional analysis of the proteolytic cleavage at the junction of the NS1 and NS2A proteins of Murray Valley encephalitis virus

Flaviviruses are a group of positive-strand RNA viruses of global significance. The flavivirus RNA genome encodes a single open reading frame that is directly translated into a single polyprotein and cleaved by host and virally encoded proteases prior to protein maturation. Hence, the proteolytic cleavage events play a central role in the process of viral gene expression in flaviviruses. Despite the importance of these events, the mechanism of proteolytic cleavage leading to the generation of the two non-structural proteins, NS1 and NS2A is poorly defined. Sequence comparisons among the flaviviruses and experimental work on Dengue virus (DENV) NS1-NS2A cleavage revealed an octapeptide sequence motif at the C-terminus of NS1 predicted to allow recognition by a protease for the cleavage of the NS1 and NS2A proteins. Of the eight-residue recognition sequence, positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A protein) are highly conserved and substitutions in these positions influenced DENV NS1-NS2A cleavage efficiency. However, the role of this recognition sequence in NS1 and NS2A production of other flaviviruses has not been experimentally addressed to date. It is also unclear whether insight gained from subgenomic expression experiments carried out in DENV is applicable to other flaviviruses. In this thesis, investigations were carried out in vitro and in vivo to assess the role of the octapeptide motif, in the efficiency of cleavage at the Murray Valley encephalitis virus (MVEV) NS1-NS2A junction. Expression cassettes encoding NS1 and NS2A genes were engineered and used for the site-directed mutagenesis of residues in the octapeptide motif. Analysis from the mutagenesis studies showed that cleavage efficiency is influenced by mutations at conserved and non-conserved residues in the octapeptides of MVEV, putatively recognized by a host protease, although overall mutations at the conserved octapeptide residues impacted more on cleavage efficiency than mutations at the non-conserved positions. Subsequently, four mutations in the octapeptide sequence (P2-Gly, P3-Gly, P8-Ala and P7,8-Ala) were introduced into an MVEV full-length infectious clone to VI investigate the impact of the substitutions during virus infection. Analysis from this study demonstrates for the first time that the efficiency of NS1-NS2A cleavage tightly controls viral RNA replication, growth in mammalian and insect cells, and virulence in mice. Despite poor conservation of amino acid at the position P2 in the octapeptide sequence, a Gin to Gly substitution at this position dramatically reduced virus replication, as demonstrated by small plaque morphology, poor RNA replication, impaired protein processing and attenuation of virulence in IFN-u{u00AC}receptor knock-out mice relative to the wild-type virus. On the contrary, non-conservative changes at highly conserved residue P3 (Val?Ala) and P8 (Leu?Ala), only slightly reduced NS1-NS2A cleavage efficiency relative to wild type and did not markedly affect virus replication. These results clearly implicate a direct association of NS1-NS2A processing with viral replication, and suggest a vital role for the octapeptide motif in modulating NS1-NS2A proteolytic cleavage. Finally, multiple growth passages of the two NS1-NS2A cleavage defective mutants, rP2-Gly and rP7,8-Ala in cell culture and mice had generated variants with revertant phenotypes. Interestingly, sequencing of the viral genome revealed that the variants had a second-site mutation in E protein in addition to the P2-Gly and rP7,8-Ala mutation at the NS1-NS2A junction. Introduction of compensatory mutation in codon 65 of E (V65A) together with the P2-Gly mutation restored the virus growth in cell culture. This result illustrates for the first time that growth deficiency of MVEV NS1-NS2A cleavage site mutants could be substantially repaired by compensatory mutations in E protein, suggesting an as-yet-unidentified role of the structural protein in NS1-NS2A cleavage or down-stream replication events. This proposition is further supported by the isolation of a putative E-NS1 polypeptide in rP2-Gly variant (containing the V65A change in E) infected cells, showing an interaction of E with NS1

Biosynthesis and Cytotoxic Activity of In Vitro Expressed Scygonadin Protein

Antimicrobial peptides (AMP) are key components of an innate immune response which represent immediate action of the defence mechanism of an organism.  It is considered a novel therapeutic agent due to its abundance in nature and a broad range of defence activity against microbial. Preceding research has shown that scygonadin AMPs isolated from seminal plasma of mud crab had the potential as a novel antimicrobial agent. However, its cytotoxicity properties on cultured cells have never been experimentally addressed. In this study, the scygonadin protein was expressed in vitro, followed by cytotoxicity assessment via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. A full-length sequence of the scygonadin gene of 387 bp was cloned into pBAD/Myc-His A vector and expressed in TOP10 cells. The protein expression was induced, purified and quantified before being subjected to cytotoxicity analysis. Next, an African green monkey kidney (Vero) cell was chosen to evaluate the cytotoxicity level of scygonadin in vitro. A total of 1x104 cells/mL were seeded into a 96-well plate before being treated to various concentrations of scygonadin protein and hydrogen peroxide as a positive control for the toxicity test.  The cells’ viability treated with scygonadin AMP and hydrogen peroxide was also verified with fluorescent analysis. The result demonstrated that the scygonadin did not cause any cytotoxicity effects while hydrogen peroxide showed an IC50 value at 0.003mM and this was further confirmed by fluorescent staining analysis. The absence of scygonadin toxicity in cells indicates its potential for biopharmaceutical use

Heterologous expression of recombinant scygonadin antimicrobial peptide from mud crab Scylla serrata

Antimicrobial peptides (AMPs) are the most common immune effectors in invertebrates that functions as the first line of defence against microbial infection. Scygonadin is an AMP which can be found in the seminal plasma of Scylla serrata. Preceding studies had shown that scygonadin have the ability to exhibit wide antimicrobial activities. Nonetheless, analysis of the antimicrobial properties of scygonadin is significantly dependent on acquiring sufficient amounts of the protein from mud crab, and this was proven difficult. Further functional studies of scygonadin and its commercial applications require a development of efficient, sustainable and cost-effective heterologous protein production. To address this issue, an expression plasmid containing 387 bp of scygonadin gene of Scylla serrata was cloned into pBAD/Myc-His A, expressed in TOP10 cells with L-arabinose as expression inducer, followed by protein purification by using immobilized metal affinity chromatography (IMAC). The optimal expression condition was determined by incubation with 0.02% of L-arabinose for 4 hours at 37°C. A total of 2 mg/ml of purified scygonadin with the molecular weight of ~17kDa was succesfully obtained. The results demonstrated that the recombinant scygonadin was successfully produced in heterologous expression system which may allow production of scygonadin in large quantities for further research and commercial application

Omics technologies used in pesticide residue detection and mitigation in crop

In agriculture, the convenience and efficacy of chemical pesticides have become inevitable to manage cultivated crop production. Here, we review the worldwide use of pesticides based on their categories, mode of actions and toxicity. Excessive use of pesticides may lead to hazardous pesticide residues in crops, causing adverse effects on human health and the environment. A wide range of high-tech-analytical methods are available to analyze pesticide residues. However, they are mostly time-consuming and inconvenient for on-site detection, calling for the development of biosensors that detect cellular changes in crops. Such new detection methods that combine biological and physicochemical knowledge may overcome the shortage in current farming to develop sustainable systems that support environmental and human health. This review also comprehensively compiles domestic pesticide residues removal tips from vegetables and fruits. Synthetic pesticide alternatives such as biopesticide and Nano pesticide are greener to the environment. However, its safety assessment for large-scale application needs careful evaluation. Lastly, we strongly call for reversions of pesticide application trends based on the changing climate, which is lacking in the current scenario

Proteolytic cleavage analysis at the Murray Valley encephalitis virus NS1-2A junction

Background: Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed. Methods and Results: Using site-directed mutagenesis at the NS1-2A cleavage site of Murray Valley encephalitis virus (MVEV), we confirmed the dominant role of conserved octapeptide residues for efficient NS1-2A cleavage, while changes at variable and the P1' residues were mostly tolerated. However, digressions from the consensus cleavage motif derived from studies on DENV were also found. Thus, comparison of the impact on cleavage of mutations at the NS1-2A junction of MVEV and DENV showed virus-specific differences at both conserved and variable residues. Conclusion: We show, with subgenomic expression and infectious clone-derived mutants of MVEV that conserved residues in the flavivirus octapeptide motif can be replaced with a different amino acid without markedly reducing cleavage efficiency of NS1 and NS2A