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HIV encapsidates viral genomic RNA and APOBEC3G in mRNA processing bodies

HIV encapsidates viral genomic RNA and APOBEC3G in mRNA processing bodies. Renato S Aguiar. Federal University of Rio de Janeiro (UFRJ) - Brazil University of California, San Francisco (UCSF) - USA. Today’s Talk. Virus RNA. Translation. Packaging. P bodies. Virus assembly

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HIV encapsidates viral genomic RNA and APOBEC3G in mRNA processing bodies

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  1. HIV encapsidates viral genomic RNA and APOBEC3G in mRNA processing bodies Renato S Aguiar Federal University of Rio de Janeiro (UFRJ) - Brazil University of California, San Francisco (UCSF) - USA

  2. Today’s Talk Virus RNA Translation Packaging P bodies • Virus assembly • Genomic RNA incorporation • mRNA processing bodies (P bodies) role

  3. Functions of unspliced viral RNA Cell cytoplasm Virus RNA  incorporation AAAA CAP translation translation translation assembly How these two different functions are coordinated?

  4. P bodies mRNA storage and degradation Newbury et al., EMBO Reports 2006 P-bodies components P-bodies components • cell development • neuron differentiation • RNA viruses mRNA storage mRNA storage Active translation Active translation APOBEC3G Staufen APOBEC3G Staufen Cellular proteins that are important for HIV replication

  5. Cellular RNA metabolism Degradation exossomes A CAP mRNA decay Stress Starvation Viral infection Storage Stress Granules Transcription P bodies AAAA CAP Translation

  6. P Bodies Knockdown of P bodies structural proteins Localize to P bodies and have been shown to function in RNA silencing. GW182 ATP-dependent RNA helicase. Rearrange RNA structures or dissociate RNA-protein complexes. RCK/p54

  7. Disruption of P bodies TZM-bl Cells – 72h postransfection GW182 knockdown RCK/p54 knockdown + + mock control siRNA GW182 siRNA + + + mock control siRNA RCK/p54 siRNA + + + + GW182 GW182 RCK/p54 actin actin WB actin RCK/p54 RCK/p54 DAPI DAPI merge merge control siRNA eIF4E-T DAPI merge control siRNA GW182 siRNA RCK/p54 siRNA Immunofluorescense

  8. siRNA transfection si c- RNA, si GW182, si RCK P Bodies role in HIV replication TZM-bl Infectivity of virus produced from P-bodies depleted cells 60h pos knockdown HIV (NL43) 5h infection wash Cell extraction luciferase assay tat tat luciferase LTR 48h pos-infection 24h pos infection Virus budded in supernatants Fresh TZM-bl cells Indicator cells 5h infection

  9. Infectivity of HIV virus produced from P bodies depleted cells TZM-bl cell lysate 100 siRNA: Control GW182 RCK + + 80 + 60 + Infectivity (%) RCK GW182 40 actin 20 p55 0 siRNA: mock control GW182 RCK p24 1 2 3 4 Reduction of HIV infectivity in virus produced from P bodies depleted cells Producer cells

  10. Does P-bodies interfere in HIV release or genomic RNA incorporation? RT-PCR (cDNA) Virus release – p24 Virus Purification Virus Real Time PCR gag region 300 Sucrose cushion 250 200 p24 levels (pg/ml) Cells 150 1.2 Virus 100 1.0 50 0.8 relative RNA levels 0 0.6 siRNA: mock control GW182 RCK 0.4 0.2 0 genomic RNA incorporation siRNA: mock control GW182 RCK

  11. P bodies are important for HIV RNA incorporation These structures can help in HIV assembly. Storage HIV genomic RNA avoiding degradation. Swanson & Malim, Traffic, 2006

  12. NEXT STEPS Binding of virus RNA and P bodies components RNA – Immunoprecipitation (similar to CHIP) HIV RNA P bodies Tag proteins Tag- Protein Ab specific to Tag-protein HIV RNA transfection clearing 293T cDNA qPCRc Beads binds Abs gag primers Release of RNA Wash-precipitation process

  13. WB-lysate a. + + + + + HIV f:Ago2 h:RCK h:eIF4E GW182.GFP + P bodies proteins binds HIV genomic RNA • Ago2 • eIF4E • RCK + + + p55 1 2 3 4 5 b. 1144 1429 LTR LTR Gag Pol Env 30 35 40 30 35 40 30 35 40 30 35 40 30 35 40 PCR cycles HIV: RNA input IP:IgG RNA IP:specific 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Gag f:Ago2 h:eIF4E h:RCK GW182.GFP Positive control

  14. Viral RNA accumulates in P Bodies Ago2.GFP HIV RNA DAPI merge Removal of P bodies disperses HIV RNA HIV RNA DAPI merge siRNA: control siRNA: RCK FISH

  15. A P body component is incorporated into new viral particles Ago2 was the only P body component to be incorporated at detectable levels into VLPs a. b. c. lymphocyte macrophage 293T cells 293T cells HIV released from primary cells incorporate Ago2 Endogenous Ago2 incorporation into viral particles is dependent of P bodies Incorporation of transient transfected Ago2 into viral particles

  16. Identification of Host Proteins Required for HIV Infection Abraham L. Brass, et al. 2008 Argonaute microRNA (miRNA) and short interfering RNA (siRNA) function

  17. GAG ALSO ACCUMULATES IN P BODIES Ago2.GFP αGag DAPI merge GW182.GFP αGag DAPI merge

  18. Structural virus protein (Gag) interacts with P bodies and this interaction is dependent of RNA Co-immunoprecipitation (Gag + P bodies proteins) Flag IgG GFP IgG HA IgG HA IgG - + - - + - - + - - + - RNAse A: IP p55 p55 p55 p55 f:Ago2 GW.GFP h:RCK h:eIF4E input p55 p55 p55 p55 Gag + Ago2 Gag + GW Gag + RCK Gag + eIF4E

  19. APOBEC3G incorporation into viral particles requires P bodies KewalRamani & Coffin, Science 2003 + + + + + HIVVif v:A3G siRNA: control GW182 RCK + + + + + + virion lysate virion lysate v:A3G v:A3G p24 p55 GW182 RCK Actin Inhibit HIV Vif replication 1 2 3 4

  20. Summary • These results are innovative and demonstrate that P bodies are required for HIV replication. • Our results suggest that these compartments are important for virus assembly and genomic RNA incorporation into new HIV particles. • Genomic RNA and Gag proteins co-localized and co-precipitated with components of P bodies, which were subsequently incorporated into new viral particles. • Depletion of P bodies also dispersed HIV genomic RNA in the cytoplasm. • Finally, these foci were also required for the incorporation of the host restriction factor APOBEC3G into progeny virions. Significance This observation suggests that interfering with P bodies could decrease HIV replication.

  21. UFRJ-Brazil UCSF-USA Acknowledgments contact: santana@biologia.ufrj.br • Matija Peterlin (UCSF-USA) • Xavier Contreras (UCSF-USA) • Amilcar Tanuri (UFRJ) • Luciana Costa (UFRJ) • Ana Luiza Valadão (UFRJ) Funding

  22. DELAYED DISAPPEARANCE OF P BODIES AFTER KNOCKDOWN OF RCK 48 h eIF4E-T 48 h 60 h 120 h siRNA: 100 60 h control 80 60 Infectivity (%) siRNA: RCK 40 20 0 RCK siRNA: control 200 180 160 Time after tranfection 140 h: 0 24 48 60 120 120 RCK 100 Infectivity (%) 80 Actin 60 1 2 3 4 5 40 RCK siRNA 20 0 siRNA: control RCK

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