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

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

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

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

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

7. Disruption of P bodies TZM-bl Cells – 72h postransfection Immunofluorescense GW182 + + + actin GW182 mock control siRNA GW182 siRNA + + + actin control siRNA RCK/p54DAPImerge RCK/p54DAPImerge GW182 siRNA mock control siRNA RCK/p54 siRNA + + + RCK/p54 actin eIF4E-TDAPImerge control siRNA RCK/p54 siRNA GW182 knockdown RCK/p54 knockdown WB

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

9. TZM-bl cell lysate Infectivity of HIV virus produced from P bodies depleted cells Producer cells Reduction of HIV infectivity in virus produced from P bodies depleted cells 0 20 40 60 80 100 Infectivity (%) mock controlGW182RCK siRNA: Control GW182 RCK ++ + + GW182 actin p55 RCK 1 2 3 4 p24

10. Does P-bodies interfere in HIV release or genomic RNA incorporation? 0 50 100 150 200 250 300 mock controlGW182RCK p24 levels (pg/ml) siRNA: Virus Purification Sucrose cushion Virus RT-PCR (cDNA) Real Time PCR gag region Virus release – p24 genomic RNA incorporation mockcontrolGW182RCK relative RNA levels 0 0.2 0.4 0.6 0.8 1.0 1.2 Cells Virus siRNA:

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

13. PCR cycles Gag f:Ago2 h:eIF4E h:RCK GW182.GFP input IP:IgG IP:specific 30 35 40 RNA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 LTR Gag Pol Env HIV: b. + + + + + HIV f:Ago2 h:RCK h:eIF4E GW182.GFP + + + + WB-lysate p55 1 2 3 4 5 a. 11441429 Positive control P bodies proteins binds HIV genomic RNA Ago2 eIF4E RCK

14. siRNA: control RCK siRNA: Viral RNA accumulates in P Bodies Removal of P bodies disperses HIV RNA HIV RNADAPImerge Ago2.GFPHIV RNADAPImerge FISH

15. a. b. c. Incorporation of transient transfected Ago2 into viral particles Endogenous Ago2 incorporation into viral particles is dependent of P bodies HIV released from primary cells incorporate Ago2 293T cells macrophage lymphocyte 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

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. Ago2.GFPαGagDAPImerge GW182.GFPαGagmergeDAPI GAG ALSO ACCUMULATES IN P BODIES

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

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

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. This observation suggests that interfering with P bodies could decrease HIV replication. Significance

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

23. Infectivity (%) 0 20 40 60 80 100 120 140 160 180 200 controlRCKsiRNA: 48 h control Infectivity (%) 0 20 40 60 80 100 siRNA: RCK siRNA:  eIF4E-T 48 h60 h control RCK 120 h siRNA: RCK 0244860120 h: Actin 1 2 3 4 5 Time after tranfection RCK siRNA DELAYED DISAPPEARANCE OF P BODIES AFTER KNOCKDOWN OF RCK 60 h