1. The role of the ubiquitin-proteasome pathway in rhTRIM5α-mediated restriction of HIV-1 Cindy Danielson Thomas Hope Laboratory, Northwestern University

2. rhTRIM5α restricts HIV-1 rhTRIM5α prevents infection of rhesus macaques by HIV-1 Sawyer 2005 Towers 2005

3. rhTRIM5α restricts HIV-1 Early post-entry block Interacts with capsid core Results in failure to complete reverse transcription Sawyer 2005 Wu 2006

4. rhTRIM5α restricts HIV-1 rhTRIM5α restriction acts prior to reverse transcription Proteasome inhibition can rescue reverse transcription while maintaining block to infection Late RT / Cell Mock HIV HIV.MG HIV.Nev % GFP+ cells HIV HIV.MG HeLa rhT5 .HeLa Wu 2006 Reverse TranscriptionInfection

5. Anderson 2006 Campbell 2008 Mechanism of Restriction Recognition of capsid core by rhTRIM5α Proteasome-sensitive block of reverse transcription Proteasome inhibitors allow visualization of intermediate stage Ubiquitin localizes to rhTRIM5α cytoplasmic bodies Campbell 2008 GFP-Vpr HA (rhTRIM5α)

6. Consequence of ubiquitination Proteasomal degradation ubiquitin rhTRIM5α Capsid ? ? ubiquitin

7. Polyubiquitination is dynamic in rhTRIM5α cytoplasmic bodies Characterizing ubiquitination +MG132

8. Characterizing ubiquitination rhTRIM5α-ΔK cytoplasmic bodies contain polyubiquitinated proteins Normalized ubiquitin/punctate HA

9. Characterizing ubiquitination Ubiquitination within rhTRIM5α-containing cytoplasmic bodies is dynamic and complex Polyubiquitination might indicate proteasomal degradation of a cytoplasmic body component

10. What is the role of the proteasome? Imaging approach using a fluorescent proteasome construct –LMP2-GFP is a β subunit of active proteasomes Tai and Schuman 2008

11. Predominantly diffuse in nucleus No treatment

12. MG132 Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with proteasome inhibition

13. Virus Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with virus

14. Proteasomes relocalize to rhTRIM5α cytoplasmic bodies Does virus interact with proteasomes? Proteasome localization # cytoplasmic proteasome puncta/cell

15. Virus associates with proteasomes Virus for 90 min No drug movie: 30 s / 30 m Long-term association can be observed

16. Virus associates with proteasomes Virus for 90 min No drug movie: 30 s / 30 m Long-term association can be observed LMP2-GFP + mCherry-Vpr

17. Virus associates with proteasomes Virus for 90 min No drug movie: 30 s / 30 m Long-term association can be observed with a decrease in virus signal

18. Virus associates with proteasomes Virus for 90 min No drug movie: 30 s / 30 m LMP2-GFP + mCherry-Vpr Long-term association can be observed with a decrease in virus signal

19. Virus associates with proteasomes Virus for 90 min No drug movie: 30 s / 30 m LMP2-GFP + mCherry-Vpr Long-term association can be observed with a decrease in virus signal

20. Virus associates with proteasomes Time course of a live cell experiment in which cells stably expressing HA-tagged rhTRIM5α were transfected with LMP2-GFP and infected with mCherry-Vpr labeled virus. Times are indicated from the start of imaging. mCherry-Vpr can be seen associated with an accumulation of LMP2-GFP for the duration of the movie (30 minutes) and the fluorescent signal of the virus appears to decrease during this association. 9 minutes 14 minutes 19 minutes 24 minutes 29 minutes LMP2-GFP mCherry-Vpr

21. Virus associates with proteasomes Proteasomes and virus associate in live cells –Stable association –Stable association with decrease in virus signal –Escape –Proteasome accumulation becomes more diffuse

22. Ongoing Studies Live cell imaging of proteasomes and rhTRIM5α Is ubiquitin recruited before proteasomes? Examining subunit composition of recruited proteasomes HA (TRIM) Rpn10 mCherry-Vpr HA (TRIM) Rpt5 mCherry-Vpr Tai and Schuman 2008

23. Conclusions Ubiquitination within rhTRIM5α cytoplasmic bodies is dynamic Proteasome localization is dynamic –Proteasomes localize to rhTRIM5α –Relocalization induced by virus or by proteasome inhibition Virus associates with proteasomes in living cells –Several types of associations can be observed –Effect of proteasome inhibition Begins to untangle the role of the ubiquitin-proteasome system in rhTRIM5α restriction of HIV-1 ubiquitin Capsid rhTRIM5α Proteasome

24. Acknowledgments Thomas J. Hope Laboratory LMP2-GFP provided by J. Neefjes Cells expressing HA-tagged rhTRIM5α provided by J. Sodroski Supported by T32 AI060523 and 5 R01 AI047770 Shannon AllenAyanna Flegler Meegan AndersonDan Gallo Ann CariasAmy Hulme Gianguido CianciAnn Koons Minh DinhMike McRaven Doug DyllaZia Okocha Carey EppesKatharina Rothwangl Alison EreksonChristopher Rold Kelly FarhbachEric Spongberg Anna FigueiredoShetha Shukair Z Kelley