Functional expression and characterization of the serina protease of the virus of the affection
Dengue (DEN) today is the most important vector-borne viral disease in tropical and subtropical countries with at least 100 million persons affected each year. It is caused by four serotypes of dengue virus (DEN-1, -2, -3, and ?4), a member of the Flavivirus genus (family Flaviviridae). Despite of these viruses have a major clinical impact, there are no vaccines, nor any specific antiviral therapeutics available for prevention and treatment of dengue infection. A serine protease encoded by the N-terminal 185 amino acids of the dengue non-structural protein 3 (NS3pro) is responsible for cleavages in cis and in trans, to generate viral proteins that are essential for viral replication and maturation. The inhibition of this process could avoid the viral replication, which makes the NS3pro an attractive target for drug development. The NS3pro requires the interaction with the 40 aminoacids NS2B core (co-factor) to be activated. A drug who competed for the protease active site could be an alternative to the blockage of the viral infection. Taking into account the success achieved with the protease inhibitors of HIV and HCV, we have decided to functionally express the serine protease complex of dengue virus (DEN1 and -2), by the co-expression of the co-factor and the protease. We have obtained success in the functional expressed of the serine protease of DEN2, showing activity when tested in synthetic chemical substrates and with similar kinetics parameters of hydrolysis to those obtained with a well-characterized DEN2 serine protease of the NGC strain. Currently, we are characterizing the DEN1 enzymatic activity. Functional protease complexes are fundamental to study the structure of the enzyme in the presence of different inhibitors, potential candidates as antiviral drugs. In addition we have engineered many chimeric protease complexes (co-factorDEN1/proteaseDEN2, co-factorDEN2/proteaseDEN1 e co-factorFA/proteaseDEN2), in order to study the specificity of the NS2B co-factor/NS3pro interaction within different DEN serotypes or even other flaviviruses. Functional studies using synthetic peptides corresponding to regions of the DEN1 and DEN2 polyproteins, which are cleaved by the protease complex, were performed. The chimeric protein DEN1co/DEN2pro showed serine protease activity for cleaving the substrates in cis as well as in trans of the DEN1 synthetic substrates. In view of these data, we wonder if the co-factor of different flavivirus could work as a target for development of a drug able to mimick NS2B and hence, interfere in the mechanism of action, by inhibiting the interaction with NS3. Currently we are testing the other chimeras with different substrates and we hope that these results could corroborate our hypothesis. In perspectives, the active complexes of protease will be used in crystallographic studies.
Advisor:Claudia Nunes Duarte dos Santos
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Date of Publication:03/04/2006