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

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
796804 Nucleic Acids Research, 2005, Vol. 33,
No. 2 doi10.1093/nar/gki220
????? ???
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INTRODUCTION

• Double-stranded RNA (dsRNA) ???
• RNA interference (RNAi),??RNAi??
• ??????????????????
• ???,HIV-1????????????
• ????RNA??????RNAi????
• ??????focus????????????
• ?????RNAi???,??????

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

• pGL3-Nef
• (firefly luciferase expression vector)
• Nef fragment
• PCR with primer EW1 and EW3
• digested with XbaI and cloned into pGL3
• pGL3-Nef

• 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-3LTR
• XhoI and BspEI fragment cloned into wild-type LAI
  clone
• R1-R9 mutant clones

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DNA constructs

• pBS-siRNA-Nef
• pRetro-SUPER-shNef vector
• (express siRNA-Nef)
• digested with EcoR1 and XhoI

H1 RNA polymerase III promoter
???
ligated into EcoR1/ XhoI site of pBluescriptII
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DNA constructs
R8 escape virus?siRNA-Nef target???????????mutant
???
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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

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CA-24 level by ELISA
Wild-type ?????RNAi??
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Materials Methods

• Cells and viruses
• DNA constructs
• Luciferase assay

• In silico RNA analysis
• RNA probing
• EMSA

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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
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Luciferase assay
????,mutant??RNAi?????
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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

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In silico RNA analysis
??R6?R8?,????????siRNA/target-RNA duplex
??????????RNAi?????(??????)
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In silico RNA analysis

• Question
• ????R6?R8????siRNA/target-
• RNA duplex ??????????RNAi
• ?????? (??????siRNA/target-
• RNA duplex ?????,??????
• ?)
• ??R8???????siRNA target??
• ?,??RNAi?????????

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In silico RNA analysis
Resistant hairpin
sensitive hairpin
Stem loop
?G??
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In silico RNA analysis
?G??
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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??,?stable ?G??,????
?? R8 R6 ???????? (R) hairpin loop??
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In silico RNA analysis

Note ??G ??0,??(R) hairpin loop? G??,????(R)
hairpin loop stable?
Mutant position at 26/-7
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DNA constructs

• m1-m4 mutants
• pGL3-Nef
• mutagenesis PCR
• with mutagenic primers EWmut1? EWmut2? EWmut3 and
  general primers EW1? EW2? EW3
• m1-m4 mutants

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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
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In silico RNA analysis
??G??,?RNAi?????????G??????(R) hairpin
loop???,????????????(R) hairpin loop?
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Materials Methods

• Cells and viruses
• DNA constructs
• Luciferase assay

• In silico RNA analysis
• RNA probing
• (Lead-induced cleavage)
• EMSA

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RNA structure probing (Lead-induced cleavage)
loop
stem
C
U
U
A
C
A
C
A
A
A
G
NoteLead-induced cleavage????????
??????????
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RNA structure probing (Lead-induced cleavage)
loop
A
A
U
C
loop
stem
G A G G
??R8?????????
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RNA structure probing (Lead-induced cleavage)

• ??Lead-induced cleavage ?????
• Mfold program ??????????
• ??
• HIV-1 RNA structure ???????
• ?RNAi ??????

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Materials Methods

• Cells and viruses
• DNA constructs
• Luciferase assay

• In silico RNA analysis
• RNA probing
• (Lead-induced cleavage)
• EMSA

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EMSA (Electrophoretic mobility shift assay)
The siRNA-Nef antisense oligonucleotide was 5
end labled in the present of 1ul of ?32PATP
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EMSA (Electrophoretic mobility shift assay)
R8 mutant bound siRNA????,free siRNA????
Note Duplex formation()
???39
bound siRNA free bound siRNA
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Conclusion

• mutant Nef gene ??????siRNA-Nef ???
• ????RNA nucleotide substitution or
  deletion?siRNA binding mismatch?
• ??luceferase assay?in silico analysis?????RNA
  probing???????????EMSA,??HIV-1 mutant???????(local
  )??RNA ?? escape RNA interference?

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Discussion

• ??RNAi??????????????
• ?????HIV-1?????????
• mutant????escape RNAi?????
• ??????????overlap??open
• reading frames ?siRNA????????
• RNAi????????????????
• ??mutant?????escape RNAi-mediated
• inhibition?

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• Thank you

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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
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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

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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

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EMSA (Electrophoretic mobility shift assay)

• ?20ul sample 2.6nm the 5-labled
  oligonucleotide
• (labled with kinaseMax kit in the present of
  ?32PATP)
• incubated 30mins, room temperature
• 4ul non-denaturing loading buffer
• Analyzed on 4 acrylamide gel
• electrophoresis