| dc.description.abstract | Japanese encephalitis virus (JEV) is a positive strand RNA virus belonging to the family Flaviviridae and genus Flavivirus. The genus includes more than 68 members, separated into groups based on serological relatedness. Flaviviruses cause serious diseases in humans and animals. Most are arthropod borne and transmitted to vertebrates by chronically infected mosquito or tick vectors. The JEV genome is approximately 11 kb long. The genomic RNA is infectious and encodes the viral proteins necessary for RNA replication. In infected cells, the genomic RNA is translated into a ~350 kDa polyprotein. The encoded protein order is: 5 -anchC–PrM–E–NS1–NS2A–NS2B–NS3–NS4A–NS4B–NS5-3 . The polyprotein is processed to individual proteins by both host signalase and a viral protease.
This thesis focuses on the nonstructural proteins NS2B and NS3 of the Indian strain P20778 of JEV. It describes:
Sequencing and sequence analysis of the NS3 gene,
Cloning and expression of the NS3 gene,
Cloning and expression of the NS2B gene, and
Demonstration of protease activity.
Sequencing and Sequence Analysis of the NS3 Gene
The complete nucleotide sequence of the NS3 gene of P20778 was determined. The NS3 gene is 1857 nucleotides long and encodes a 619 amino acid protein. Comparison of P20778 NS3 with that of the Japanese strain JaoArS982 revealed 95.9% nucleotide identity. Restriction site analysis indicated the appearance of eight new sites (AsuII, AvaI, BglII, NheI, two MscI, SacI, PstI) and the loss of five sites (AccI, two BbvII, HgiAI, Tth111I) in P20778.
At the amino acid level, P20778 showed 98.38% identity with JaoArS982. Notable substitutions in P20778 occurred at positions 398 (K E), 402 (D A)-converting a negatively charged residue to non polar-and 467 (N K)-introducing a positive charge. The calculated pI values were 7.32 (P20778) and 7.12 (JaoArS982).
The N terminal 184 amino acids of NS3 (P20778) revealed significant homology to cellular serine proteases in four regions. H51, D75, and S135 constitute the catalytic triad of NS3 protease. The conserved motif G X S G X P represents the nucleophilic serine region of the protease. The conserved D129 followed by Y/F/L, and the motif G L Y G N G, are hypothesized to form part of the substrate binding pocket influencing cleavage specificity.
Cloning and Expression of the NS3 Gene
The P20778 NS3 gene was cloned into the T7 promoter based vector pET3d. The initial recombinant was non productive, due to an inhibitory stem–loop RNA secondary structure encompassing nucleotides 3–30 of the NS3 coding sequence (predicted G = 13.4 kcal/mol) that impeded translation initiation.
Given that NS3 protease activity resides within the N terminal 184 aa, the amino acids encoded by this structured region may be essential for proper folding and activity. A novel expression strategy was devised:
An upstream AUG was introduced before the structured region,
A 12 nt spacer between the new AUG and the stem–loop provided the footprint for the initiating ribosome,
A second codon for glycine was placed to facilitate initiator methionine removal.
Using this design, the NS3 gene was successfully expressed in pET3d.
Initiation at the upstream AUG was confirmed by N terminal protein sequencing. Deletion of the stem–loop in the non productive construct yielded expression of a truncated NS3, confirming that the secondary structure inhibited expression. Northern blotting detected NS3 specific mRNA in E. coli harboring both productive and non productive constructs. The protease domain of NS3 (NS3T) was expressed in pET3d and validated by western blotting.
Cloning and Expression of the NS2B Gene
NS2B is required for NS3 protease activity. To study proteolysis, NS2B expression was essential. NS2B was first expressed as a GST–NS2B fusion (vector pET3d) and purified on glutathione–agarose. In subsequent transformations/inductions, only GST was obtained. NS2B was therefore expressed as a NS3T–NS2B fusion, enabling in vitro protease assays.
Protease Activity
A fluorogenic peptide substrate (Dns NKKRGWPA) for NS3T–NS2B was synthesized using Fmoc solid phase peptide synthesis. NS3T–NS2B localized to inclusion bodies when expressed in E. coli (M9 medium at 20°C or 30°C), yielding insoluble protein. Inclusion bodies were isolated, partially purified, and solubilized in 8 M urea/6 M guanidinium HCl. Refolding attempts (stepwise dialysis, stepwise dilution, with/without glutathione redox) did not yield active enzyme.
An alternate strategy was used to demonstrate proteolysis in vivo: a recombinant polyprotein NS3T–NS2A–NS2B was engineered, where NS3T is the protease and NS2B its cofactor; the natural NS2A/NS2B junction served as substrate. Upon IPTG induction, the polyprotein underwent self cleavage, generating a ~47 kDa cleavage product-demonstrating protease activity.
Summary of Results
The complete NS3 gene sequence (1857 nt) of P20778 was determined.
The NS3 gene was expressed in a prokaryotic system using a novel upstream AUG strategy to bypass an inhibitory stem–loop.
The NS2B gene was expressed as an NS3T–NS2B fusion.
The proteolytic activity of the NS3T–NS2B protease was demonstrated.
Conclusions and Implications
Although immunological methods exist for JEV detection, a more sensitive RT PCR approach using NS3 consensus region primers could improve speed and sensitivity in clinical diagnostics. The novel expression strategy developed here can be applied to other genes whose translation is hindered by 5 mRNA secondary structures; its effectiveness was validated by demonstrating protease activity of the P20778 enzyme. This study provides the first demonstration of activity for bacterially expressed JEV protease. Further structural and functional analyses of the protease may aid antiviral drug development against JEV. | |
