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HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Ellen M. Westerhout, Marcel Ooms, Monique Vink, Atze T. Das and Ben Berkhout*.

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HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

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  1. HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome Ellen M. Westerhout, Marcel Ooms, Monique Vink, Atze T. Das and Ben Berkhout* Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Received December 10, 2004; Revised and Accepted January 13, 2005 796–804 Nucleic Acids Research, 2005, Vol. 33, No. 2 doi:10.1093/nar/gki220 生科系四甲 張智堯

  2. INTRODUCTION Double-stranded RNA (dsRNA) 能誘導 RNA interference (RNAi)，利用RNAi的技 術可以有效的抑制基因的表現。而在研究 中發現，HIV-1病毒中卻有突變種可以藉由 改變自己RNA的結構來降低RNAi抑制基因 的效果。因此focus在這些突變種的改變結構為 何能夠抵抗RNAi的抑制，並加以驗證。

  3. RNA interference (RNAi) a ribonuclease short interference RNA

  4. RNA interference (RNAi)

  5. HIV

  6. HIV genome

  7. Materials & Methods Cells and viruses DNA constructs Luciferase assay In silico RNA analysis RNA probing EMSA

  8. Cells and viruses • C33A cervix carcinoma cell calcium phosphate method 5 ug wild-type or mutant HIV-1 LAI transfection

  9. Materials & Methods Cells and viruses DNA constructs Luciferase assay In silico RNA analysis RNA probing EMSA

  10. DNA constructs • R1-R9 mutants： cellular DNA PCR with 5’ Env primer tTAI-AD and 3’ U5 primer CN1 digested with XhoI and BspEI and cloned into Blue-3’LTR XhoI and BspEI fragment cloned into wild-type LAI clone R1-R9 mutant clones • pGL3-Nef (firefly luciferase expression vector)： Nef fragment PCR with primer EW1 and EW3 digested with XbaI and cloned into pGL3 pGL3-Nef

  11. DNA constructs • pBS-siRNA-Nef： pRetro-SUPER-shNef vector (express siRNA-Nef) digested with EcoR1 and XhoI H1 RNA polymerase III promoter 建構成 ligated into EcoR1/ XhoI site of pBluescriptII

  12. DNA constructs R8 escape virus在siRNA-Nef target區域部分卻沒有沒有產生mutant ???

  13. CA-24 level by ELISA • SupT1 T-cells transduced with pRetro-SUPER cultured in RPMI 1640 medium. HIV-1 LAI infect (1ng of CA-p24) CA-24 level by ELISA

  14. CA-24 level by ELISA Wild-type 如預期的被RNAi抑制

  15. Materials & Methods Cells and viruses DNA constructs Luciferase assay In silico RNA analysis RNA probing EMSA

  16. Luciferase assay 100 ng wild-type or mutant pGL3-Nef + 0.5ng pRL-CMV + 0.5-500 ng pBS-siRNA-Nef completed with pBluescriptII 1ug (total), 15ul water Mixed 25ul 2XHBS, 10ul 0.6MCaCl2 to cultrue medium, incubated room temperature 20 mins refreshed 16h 24h lysed in 150ul Passive Lysis Buffer (PLB) shaking 20 mins at room temperature mixed centrifuged 10ul supernatant to measure luciferase

  17. Luciferase assay 總體來說，mutant皆對RNAi有抵抗作用

  18. Materials & Methods Cells and viruses DNA constructs Luciferase assay In silico RNA analysis RNA probing EMSA

  19. In silico RNA analysis Mfold program • Hybridization can be predicted • Thermodynamic stability (△G) can be caculated • RNA structures can be predicted

  20. In silico RNA analysis 除了R6與R8外，所有突變種皆隨著siRNA/target-RNA duplex穩定性的減少而增加對RNAi的抵抗力。(趨近線性關係)

  21. In silico RNA analysis Question 為何只有R6與R8沒有隨著siRNA/target- RNA duplex穩定性的減少而增加對RNAi 的抵抗力?? (事實上他們的siRNA/target- RNA duplex穩定性較高，卻有較高抵抗 力) 尤其R8的突變位置不在siRNA target的區 域，卻對RNAi有較高的抵抗力??

  22. In silico RNA analysis Resistant hairpin sensitive hairpin Stem loop △G上升

  23. In silico RNA analysis △G下降

  24. In silico RNA analysis R8 mutant：(S) Hairpin less stable -21.3 -17.9 -15.0 -17.9 R6 mutant：(R) Hairpin more stable -15.1 -13.0 Note： △G越小，越stable； △G越大，越不穩定 所以 R8 & R6 因此形成較穩定的 (R) hairpin loop結構

  25. In silico RNA analysis = Note： △△G 大於0，表示(R) hairpin loop△ G較低，所以其較(R) hairpin loop stable。 Mutant position at –26/-7

  26. DNA constructs • m1-m4 mutants： pGL3-Nef mutagenesis PCR with mutagenic primers EWmut1、 EWmut2、 EWmut3 and general primers EW1、 EW2、 EW3 m1-m4 mutants

  27. Luciferase assay Note： m1 △△G = 6.8 R8 △△G = 2.0 m2 △△G = -2.4 m3 △△G = -2.8 m4 △△G = -3.5 wt △△G = -2.8

  28. In silico RNA analysis △△G越高，對RNAi的抵抗力越強；而△△G越高也表示其(R)hairpin loop越穩定，會使結構趨向形成較穩定的(R) hairpin loop。

  29. Materials & Methods Cells and viruses DNA constructs Luciferase assay In silico RNA analysis RNA probing (Lead-induced cleavage) EMSA

  30. RNA structure probing (Lead-induced cleavage) stem loop C U U A C A C A A A G Note：Lead-induced cleavage主要是切單股結構 所以與預測結構相符合

  31. RNA structure probing (Lead-induced cleavage) loop A A U C loop stem G A G G 所以R8也與預測結構相符合

  32. RNA structure probing (Lead-induced cleavage) • 經由Lead-induced cleavage 可以證明由 Mfold program預測出來的結構是正確 的。 • HIV-1 RNA structure 的改變確實避開 了RNAi 作用的區域。

  33. Materials & Methods Cells and viruses DNA constructs Luciferase assay In silico RNA analysis RNA probing (Lead-induced cleavage) EMSA

  34. EMSA(Electrophoretic mobility shift assay) The siRNA-Nef antisense oligonucleotide was 5’ end labled in the present of 1ul of [γ32P]ATP

  35. EMSA(Electrophoretic mobility shift assay) R8 mutant bound siRNA明顯較少，free siRNA明顯較多 Note： Duplex formation(%) 不超過39% bound siRNA = free + bound siRNA

  36. Conclusion • mutant Nef gene 確實可以抵抗siRNA-Nef 作用。 • 可以藉由RNA nucleotide substitution or deletion使siRNA binding mismatch。 • 經由luceferase assay、in silico analysis預測結構、RNA probing證實預測結構的正確性、EMSA，證明HIV-1 mutant型確實藉由局部(local)改變RNA 結構 escape RNA interference。

  37. Discussion 經由RNAi這個強大的基因治療工具雖然確 實可以抑制HIV-1的病毒複製。但是其 mutant型卻可以escape RNAi的抑制作用； 或許可以設計一個可以overlap整個open reading frames 的siRNA來使病毒無從閃躲 RNAi的抑制作用。在藥物設計上也要開始 考量mutant型的病毒會escape RNAi-mediated inhibition。

  38. Thank you

  39. RNA structure probing (Lead-induced cleavage) wild type and R8 (20pmol) denature 60ul water 85 ℃ 3mins snap cooling on ice 20ul 4XMO buffer incubated 30 mins 37 ℃ incubated with lead(II) acetate at room temperature stop cleavage by 3ul 1M EDTA Samples (15ul) 0, 5, 15, 25 mins

  40. RNA structure probing (Lead-induced cleavage) 3pmol 32p-labled oligonucleotide was annealed to 3pmol of the lead(II)-treated RNA by incubation at 85 ℃ 3mins slow cooling 60 ℃ 1h 20ul gel-loading bufferII samples heated 95℃ 10ul samples analized on denatured 6% arylamide

  41. EMSA(Electrophoretic mobility shift assay) wild type and R8 denature in 30ul water at 85℃3 mins snap cooling renatured with 10ul 4X MO buffer 37 ℃ 30mins the transcripts were diluted in 1X MO buffer final concentration 0 to 7.5 M in MO buffer

  42. EMSA(Electrophoretic mobility shift assay) 取20ul sample + 2.6nm the 5’-labled oligonucleotide (labled with kinaseMax kit in the present of [γ32P]ATP) incubated 30mins, room temperature 4ul non-denaturing loading buffer Analyzed on 4% acrylamide gel electrophoresis

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