1. “Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1” Ihtisham Ul Haq Roll No: 13 Subject: Seminar 5 th Semester, Department of Microbiology Abdul Wali Khan University Mardan

2. Introduction The dengue virus – a flavivirus of which there are four serotypes. Dengue is widespread in tropical and subtropical regions of central and South America and south and south-east Asia. They translates on the host cell ribosomal machinery to produce polypeptide. NS3 protein is one of the most important non-structural proteins. The activity of NS3pro is depends on the interaction with NS2B protein to form NS2B-NS3pro. NS2B-NS3pro is considered as potential target for antiviral drug design. Retrocyclin-1 is disulphide cyclic peptide which has significantly inhibitory potential against Dengue NS2B-NS3pro.

3. Construction of plasmids pQE30-NS2BNS3pro Mosquito cell line C6/36 infected with DEN-2 viruses and incubated at 32°C for 3 days. For amplification cDNA fragments of NS2B-NS3pro, viral RNA was extracted using viral RNA Extraction Kit based onto the manufacturer’s instructions The cDNA fragments was generated using NS2BF and NS3R. These sequences then used as template to amplify NS2B and NS3pro by PCR reactions The linker sequence and KpnI restriction sites were added to NS2BlikR and NS3likR primers. The PCR products were subsequently digested with BamHI and KpnI. The purified fragments were cloned into pQE30 plasmid downstream of the 6XHis tag.

4. Protein production in E.coli

6. Antimicrobial activity of RC-1 The disc diffusion susceptibility test was used to evaluate antimicrobial activity of the recombinant RC-1. The samples of recombinant RC-1 were dropped onto a filter paper discs (0.1 mg/ml). Then, the filter paper discs placed on E.coli inoculated MH agar and Saccharomyces cerevisiae inoculated YPD agar plates. Both inoculated plates were inoculated for 16 hrs at 35°C and 30°C, respectively.

8. Treatment of DENV-2 infected cells with recombinant RC-1 Three independent experiments were carried out with RC-1 at 24, 48 and 72 hrs of each infection in triplicates which is:  Pre-infection treatment  Simultaneous infection treatment  Post-infection treatment Vero cells were grown in a 24-well tissue culture plate (1×105 cells/well). Incubated for 24 hrs under optimal conditions and infected with DENV-2.

10. Result: The inhibitory potential of RC-1 against Dengue protease The ability of NS2B-NS3pro to cleave the fluorogenic peptide substrate was variable at different temperatures  Highest activity at 28°C  Lowest activity at 40°C  Midrange activity at 37°C The inhibition profile of RC-1 was variable at different temperatures  At 28°C, RC-1 exhibited 100% inhibition of NS2B-NS3pro at concentration of 120 μM  Complete inhibition was observed at 100 μM and 37.5 μM at 37°C and 40 °C  The lowest 50% inhibitory concentration (IC50 value) of RC-1 was observed at 40°C (14.1 ± 1.2 μM) and the highest IC50 at 28°C (46.1 ±1.7 μM) whilst at 37°C, the IC50 of RC-1 was 21.4 ± 1.6 μM.

13. The inhibitory potential of RC-1 against dengue virus replication in Vero cells The maximum non-toxic dose (MNTD) of RC-1 concentration in Vero cells was found to be 150 μM for all of the three incubation periods, 24, 48 and 72 hrs. RNA of DENV-2 was extracted from RC-1 treated and control cells and quantified by real-time PCR. The results showed that the highest percentage of reduction in total viral RNA copies was observed for the simultaneous treatment modes as compared to pre and post treatment modes.

14. Conclusion We successfully produced bioactive recombinant RC-1 in E. coli as a cost-effective expression system that can be developed for large-scale production. The recombinant RC-1 was able to inhibit dengue protease that led to reduction of dengue virus in Vero cells. These findings may have its importance in the development of therapeutic agent especially against dengue infection.