1 / 17

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

Dimerization of differentially phosphorylated Hepatitis C Virus non-structural protein 5A (NS5A). JULIAN AVILA. Hepatitis C Virus. Affects about 3% of the world’s population, 80% of the infections become chronic. Cirrhosis and cancer. Limited treatments. Hepatitis C Virus. ss(+) RNA.

india-osborn
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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]

More Related