Summer Research Inhibition of Type I Interferon Signal Transduction Pathways by JEV infection

1. Summer Research Inhibition of Type I Interferon Signal Transduction Pathways by JEV infection 中央研究院生物醫學科學研究所 林宜玲 老師 University of California, Los Angeles Summer Student: 陳勃睿 清華大學生科05級 2003/12/5

2. Introduction • Flaviviruses Journal of Infection (2001) 42, 104–115

3. Introduction • Flaviviruses • Enveloped virus • Single-strand, positive sense RNA as genome • One ORF encoding a polyprotein * * NS1 C E NS3 NS5 NS2B NS2A prM/M NS4A NS4B : host signalase : viral protease NS2B-3 : furin or other golgi-localized protease : unknown protease : glycosylation *

4. Introduction • Interferons (IFNs) are a family of cytokines that proceed antiviral activity. -Type I (viral IFNs) • include IFN-α, IFN-β, IFN-ω, IFN-τ • produced by most body cells -Type II (immune IFNs) • include IFN-γ • produced by certain immune cells only such as CD4+ T help cells

5. Introduction • Signal Transduction Pathways

6. Introduction • Viral interference in the IFN system

7. Material • Japanese Encephalitis Virus - E domain II, prM/M, E • pIRES-hrGFP, r21-TE12Q • Competent Cells: Top10, DH5α • Transfection: BHK-21 (mouse)

8. Method • Molecular Cloning Technique • Transfection • Immunofluorescence Assay (IFA) • Western Blotting • Polymerse Chain Reaction (PCR) • Deletion with one primer in single step

9. Molecular Cloning 養菌，抽 plasmid JE-II/pSecTag B pIRES-hrGFP Nhe I Sma I T4 DNA polymerase Sal I Sal I CIP (AP) Gel Extraction Ligation Transformation

10. Transfection • Lipofectamine • artificial membrane • Sindbis Virus • interferon-sensitive virus

11. Immunofluorescence Assay DAPI

12. Immunofluorescence Assay DAPI

13. Western Blotting • Failed...

14. Single Primer Deletion • higher efficiency than primer pairs (although less colonies) • DpnI (5’-Gm6ATA-3’) • cost is reduced BioTechniques Vol.29, No.5, 970-972

15. Lab Members

16. Thank you!