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HIV-1 Alternative splicing

Both linear and discontinuous ribosome scanning are used for translation initiation from bicistronic HIV-1 env mRNAs.

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HIV-1 Alternative splicing

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  1. Both linear and discontinuous ribosome scanning are used for translation initiation from bicistronic HIV-1 env mRNAs. Adam T. Johnson1, Jenny L. Anderson1,Jane L. Howard1, Jörg Krummheuer2, Ilona Hauber3, Susanne Kammler1, Joachim Hauber3, Heiner Schaal2and Damian F. J. Purcell1 1University of Melbourne, Microbiology and Immunology, Parkville Australia, 2Heinrich-Heine-University, Institute for Virology, Dusseldorf, Germany, 3Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany

  2. env poly(A) 3’ poly(A) 3’ 5’ Cap 5’ Cap Spliced mRNA 9kb Unspliced pol gag HIV-1 Alternative splicing 9kb genome rev tat pol gag rev tat

  3. env env poly(A) 3’ poly(A) 3’ 5’ Cap 5’ Cap Spliced mRNA 9kb Unspliced pol gag SA4b SD1 5’ Cap HIV-1 Alternative splicing 9kb genome rev tat pol gag rev tat 4kb Singly spliced poly(A) 3’

  4. env env poly(A) 3’ poly(A) 3’ 5’ Cap 5’ Cap Spliced mRNA 9kb Unspliced pol gag 5’ Cap 5’ Cap SD4 SA7 poly(A) 3’ HIV-1 Alternative splicing 9kb genome rev tat pol gag rev tat 4kb Singly spliced poly(A) 3’ 2kb Multiply spliced rev

  5. 5 4b SA: 7 2 3 4 6 4a 4c 2 3 4 5 SD: env mRNAs % abundance 5E env 1 1 / 5E env 2 1 / 4bE 51 10 4bE env 3 1 / 4aE env 4 1 / 4cE env 5 1 / 2 / 5E env 6 1 / 2 / 4bE env 7 1 / 2 / 4aE env 9 1 / 2 / 4cE env 8 1 / 3 / 5E env 10 1 / 3 / 4bE env 11 1 / 3 / 4aE env 12 1 / 3 / 4cE env 13 1 / 2 / 3 / 5E env 14 1 / 2 / 3 / 4bE env 15 1 / 2 / 3 / 4aE env 16 1 / 2 / 3 / 4cE 11 3 6 1 2 <1 8 2 2 <1 3 <1 <1 <1 4aE 4cE 5E 4bE 4aE 4cE 5E 4bE 4aE 4cE 5E 4bE 4aE 4cE HIV-1 generates 16 env mRNAs that differ in their 5’ UTR Primary mRNA env Cap AAA 1 vpu

  6. PABP Rev PABP eIF2 La tRNAlys3 TRBP PKR Cyclin T Tat ORFs (All env mRNAs) HIV-1 env mRNAs: RNA elements, structures and RNA-protein interactions TAR RRE Poly A rev1 TAR AA(A)n 7mGpppN env PBS DIS nef vpu Exon 1 Exon 3 Exon 2 5’ UTR

  7. - + + + rev AUG SA 4c 4a 4b 5 nef vpu rev 2 rev 1 M. MOCK e1. 1/5E e2. 1/4bE e3. 1/4aE e4. 1/4cE e5. 1/2/5E e6. 1/2/4bE e7. 1/2/4aE e8. 1/3/5E e10. 1/3/4bE e11. 1/3/4aE e13. 1/2/3/5E e14. 1/2/3/4bE env Ex 2? Ex 3? Ribosome Shunt Weak kozak Strong kozak Translation controlled by “leaky ribosome scanning”? - - - + - + + + + + + + rev AUG M e1 e2 e3 e4 e5 e6 e7 e8e10 e11 e13 e14 Env Vpu Alternative env mRNAs code gp160 with equal efficiency, but Vpu with different efficiency.

  8. Ribosome Shunting G/ACCAUGG AUG uORF AAA m7G Requires RNA secondary structures to act as a launching & landing pad Shunting elements may also require the presence of a short uORF to position the 40S ribosome at the correct site for shunting

  9. RRE 4b 5 SA nef vpu rev 2 m7G cap AAA Env (E) rev 1 env egfp Strong Kozak AUG RRE 4b 5 SA Weak Kozak AUG nef vpu rev 2 R38 Mutation m7G cap EnvGFP (EG) AAA rev 1 env Env expression and reporter constructs

  10. Env1 (1/5E) Env2 (1/4bE) Env (E) EnvGFP (EG) Env-GFP expressed as a chimeric fusion protein E1RG E2RG Mock E1R E2R FL-GFP gp160 (FL) TM-GFP gp41 (TM) Env mAb C8 GFP

  11. E2RG E1RG E2(env)RG E1(env)RG E2R E1R Mock 0 100 200 300 400 500 600 700 800 Relative Fluorescence Expression of GFP is a measure of translation initiation at the env AUG sj 1.4b env gfp vpu LTR sj 1.5 env gfp vpu LTR sj 1.4b  env gfp vpu LTR sj 1.5  env gfp vpu LTR sj 1.4b env vpu LTR sj 1.5 env vpu LTR

  12. Conservation of a uORF within the 5’UTR of env mRNA’s SD4 uORF Vpu pNL4-3 CAAAGCAGTAA..........GTAGTACATGTAATGcAACCT...............ATA CONSENSUS_01_AE -----------GTA......ATA----T--------tC----...............T-GCONSENSUS_A1+A2(15) -----------GTATTAgTAAT-----T--------aCT---...............T-- CONSENSUS_B(33) -----------..........------------------T--...............T-- CONSENSUS_C(41) -----------.............---T--------tT-GA-ttactagcaaaagtaGAT CONSENSUS_D(8) -----------..........---------------------...............T-- CONSENSUS_F1+F2(6) -----------...GTGtTAA------T--------tC-TA-...............T-G CONSENSUS_G(5) -----------GTAGTAaTAAT-----T-------------A...............T-- CONSENSUS_H(3) -----------.............---TC-T-----.....................--- J.SE.SE91733 --G--------GTA.ACtTAAT---C-T--------gT-T--...............T-- K.CM.MP535C G--------G-GTAGTGcTAA---------------gTGT-C...............T-- O.CM.ANT70 -----------.............---ACGC-------T-A-...............-GG 16nt between SD4 and the Vpu AUG is the longest region of non-coding sequence within HIV-1 genome Does this conserved uORF play a role in maintaining consistent Env levels?

  13. sj 1.5 env gfp LTR vpu vpu uORF E1RG UAGUACAUGUAAUGC E1eRG UAGUACAUGCAAUGCaacctaUAA E1sRG UAGUACAAGUAAUGC E1RG E1eRG E1sRG 0 20 40 60 80 100 120 Vpu Env-Expression [%] Vpu-Expression [%] 0 25 50 75 100 125 Disrupting a minimal uORF reduces the level of Env expression

  14. sj 1.5 env gfp LTR vpu vpu uORF E1RG UAGUACAUGUAAUGC E1(VK)RG UAGUACAUGUAAUGG E1(VT)RG UAGUACAAGAAAUGG -3 +1 +4 E1RG E1(VK)RG E1(VT)RG 0 50 100 150 200 0 20 40 60 80 100 120 Vpu Vpu-Expression [%] Env-Expression [%] Increasing the strength of the vpu initiation codon further reduces the levels of Env expression Vpu Kozak Strength +/- ++ +++

  15. sj 1.4b sj 1.4b env env gfp gfp LTR LTR vpu vpu vpu uORF vpu uORF E2RG UAGUACAUGUAAUGC E2RG UAGUACAUGUAAUGC E2RG E2eRG UAGUACAUGCAAUGCaacctaUAA E2(VK)RG UAGUACAUGUAAUGG E2sRG UAGUACAAGUAAUGC E2(VK)RG E2(VT)RG UAGUACAAGAAAUGG -3 +1 +4 E2(VT)RG E2RG E2eRG E2sRG 100 120 40 60 80 0 20 Env-Expression [%] 0 20 40 60 80 100 120 Env-Expression [%] An upstream Rev AUG alleviates the effect of uORF mutations on Env Expression

  16. Summary • Env is translated from the downstream env ORF using both • an unusual discontinuous translation mechanism, and • a linear reinitiation mechanism • Mutations that extend or remove an short uORF before the authentic Vpu start strongly inhibit Env expression • This uORF may pause the 40S ribosome at an RNA secondary structure responsible for ribosome shunting. • Recognition of the uORF requires initiation competent 40S ribosomes. • An upstream rev ORF reduces the interaction with uORF before Vpu. • Novel translation mechanisms suggests Env is a candidate for translation modulation that could impact upon viral infectivity • Cell cycle, cell stress, miRNA can modulate translation of select mRNA.

  17. Acknowledgements Adam Johnson Jenny Anderson Jane Howard Marina Alexander Heiner Schaal Jörg Krummheuer Ilona Hauber Susanne Kammler Joachim Hauber

  18. - + + + rev AUG SA 4c 4a 4b 5 nef vpu rev 2 R38 rev 1 20 env 15 Fold CAT Activation above Mock 10 5 0 gp160 M NL E2R E3R E4R E2R E3R E1R E1R E4R Rev Protein expression from env mRNA isoforms mutated to equalise for Rev activity (R38)

  19. SA5 SA4b SD4 exon 2 exon 1 rev1 env m7G vpu wt: AGU UUG GAA AGG ACC AGC AAA KAGG: AGU GGC ACC AGG GCC AGC UAA KAUG: AGU GGC ACC AUG GCC AGC UAA stop start 140 120 120 100 100 80 80 Env Expression [%] Vpu Expression [%] 60 60 40 40 20 20 0 E5 E6 E5KAGG E6KAGG E5KAUG E6KAUG 0 E5 E6 E5KAGG E6KAGG E5KAUG E6KAUG gp160 Vpu Rev Rev Inclusion of a strong AUG in non-coding exon 2 does not effect Env expression

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