Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1
Background; Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2012
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| Subjects: | |
| Online Access: | http://eprints.um.edu.my/7127/1/1471-2334-12-314.pdf http://eprints.um.edu.my/7127/ |
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| Summary: | Background; Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro. Methods: Dengue NS2B-NS3pro was produced as a recombinant single chain protein in E. coli and purified by Ni+ affinity chromatography. The RC-1 peptide was produced in E. coli and the tri-disulphide bonds were reformed in a diluted alkaline environment. Protease assay was performed using a fluorogenic peptide substrate and measured by fluorescence spectrometry. Real-time PCR was used for quantification of dengue serotype 2 (DENV-2) viral RNA produced in Vero cells. Results: The RC-1 peptide inhibited the activity of recombinant NS2B-NS3pro with different values at 50 inhibitory concentration (IC50) which are temperature dependent (28°C, 46.1�±�1.7 μM; 37°C, 21.4�±�1.6 μM; 40°C, 14.1�±�1.2 μM). The presence of RC-1 significantly reduced viral replication in Vero cells infected with DENV-2 at simultaneous treatment after 48 hrs (70) and 75 hrs (85). Furthermore, moderate reduction in viral replication was observed at pre-treatment mode after 48 hrs (40) and 72 hrs (38) and post-treatment at 48 hrs (30) and 72 hrs (45). Conclusion: Recombinant RC-1 inhibits DENV-2 replication in Vero cells by interfering with the activity of its serine protease. Thus, we propose that recombinant RC-1 is a potent, cost-effective dengue virus inhibitor. Therefore, it is suitable to consider RC-1 as a new candidate for drug development against dengue infection. |
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