Dimerization of differentially phosphorylated Hepatitis C Virus non-structural protein 5A (NS5A) - PowerPoint PPT Presentation

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Dimerization of differentially phosphorylated Hepatitis C Virus non-structural protein 5A (NS5A)

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Dimerization of differentially phosphorylated Hepatitis C Virus non-structural protein 5A (NS5A)
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Dimerization of differentially phosphorylated Hepatitis C Virus non-structural protein 5A (NS5A)

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  1. Dimerization of differentially phosphorylated Hepatitis C Virus non-structural protein 5A (NS5A) JULIAN AVILA

  2. Hepatitis C Virus • Affects about 3% of the world’s population, 80% of the infections become chronic. • Cirrhosis and cancer. • Limited treatments.

  3. Hepatitis C Virus ss(+) RNA 5’ NTR 3’NTR ± 9600nt Flaviviridae

  4. NS5A 36 198 447 (2419) 1 (1973) Tellinghuisen et al., Nature (2005)435:375-379

  5. NS5A BASAL PHOSPHORYLATION (2200-2250) BASAL PHOSPHORYLATION (2350-2419) 447 (2419) 1 (1973) 49KDa HYPERPHOSPHORYLATION (S2197, S2201, S2204) REPLICON SYSTEM 32P labeling Interferes with RNA replication HIGH VIRAL RNA REPLICATION.

  6. Hypothesis Hyperphosphorylation of NS5A interferes with its dimerization. ? BASALLY PHOSPHORYALATED DIMERIC REPLICATING FORM HYPERPHOSPHORYLATED

  7. First specific Aim Does hyperphosphorylation affect NS5A dimerization in vitro? Express NS5A-His in E. coli (wt and NS5A S2204I) Phosphorylate in vitro using tagged zebrafishCKI-α expressed and purified in E. coliand [32P]-γ-ATP.

  8. Does hyperphosphorylation affect NS5A dimerization in vitro? NS5A-HIS NS5A-S2204I p58 p56 SDS PAGE CKI-α - - + + - - + + 32P-ATP - + - + - + - + Phosphorimaging p58 p56 p58 p56 * S2204I

  9. Equilibrium sedimentation 49 40 μM 15,000 r.p.m. 12,000 r.p.m. A (280 nm) 8,000 r.p.m. radius (cm)

  10. p58 p56 S2204I Equilibrium sedimentation 40 μM 20 μM 4 μM 15,000 r.p.m. 12,000 r.p.m. A (280 nm) 8,000 r.p.m. radius (cm) radius (cm) radius (cm)

  11. Equilibrium sedimentation * 40 μM 20 μM 4 μM 15,000 r.p.m. 12,000 r.p.m. A (280 nm) 8,000 r.p.m. radius (cm) radius (cm) radius (cm)

  12. Second specific aim In vivo interaction of phosphorylated NS5A + Huh-7 cells S2204I 1 Jc1 - NS5A-RFP Jc1- NS5A S2204I-GFP-His S2204I 1 Adapted from Schaller et al., (2007) J Virol. 81:4591-4603

  13. Second specific aim In vivo interaction of phosphorylated NS5A 1 Huh-7 cells 1 Jc1 - NS5A-RFP Jc1- NS5A S2204I-GFP-His S2204I 1 Adapted from Schaller et al., (2007) J Virol. 81:4591-4603

  14. Jc1- NS5A S2204I-RFP-His S2204I Precipitated Precipitated wce wce In vivo interaction of phosphorylated NS5A 85 83 α-RFP 85 83 α-GFP S2204I S2204I S2204I S2204I 1 Jc1 - NS5A-RFP Jc1- NS5A S2204I-GFP-His S2204I 1 Adapted from Schaller et al., (2007) J Virol. 81:4591-4603

  15. Future directions • Identify domains necessary for NS5A dimerization. • Evaluate impact of phosphorylation of NS5A on RNA binding. • Evaluate mobility of NS5A and cellular localization of phosphorylated forms. • Investigate differential binding of phosphoforms of NS5A to viral and cellular proteins.

  16. Quintavalle et al., 2006 Schaller, et al. 2007

  17. Sednterp estimates Using >gi|29365458|gb|AAO53246.1| non-structural 5A [Hepatitis C virus]