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The role of the ubiquitin-proteasome pathway in rhTRIM5α-mediated restriction of HIV-1

The role of the ubiquitin-proteasome pathway in rhTRIM5α-mediated restriction of HIV-1. Cindy Danielson Thomas Hope Laboratory, Northwestern University. rhTRIM5 α restricts HIV-1. rhTRIM5α prevents infection of rhesus macaques by HIV-1. Sawyer 2005. Towers 2005. rhTRIM5 α restricts HIV-1.

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The role of the ubiquitin-proteasome pathway in rhTRIM5α-mediated restriction of HIV-1

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  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 Wu 2006 Sawyer 2005

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

  5. GFP-Vpr HA (rhTRIM5α) Campbell 2008 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

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

  7. Characterizing ubiquitination • Polyubiquitination is dynamic in rhTRIM5α cytoplasmic bodies +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. Tai and Schuman 2008 What is the role of the proteasome? • Imaging approach using a fluorescent proteasome construct • LMP2-GFP is a βsubunit of active proteasomes

  11. No treatment • Predominantly diffuse in nucleus

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

  13. Virus • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with virus

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

  15. Virus associates with proteasomes • Long-term association can be observed Virus for 90 minNo drugmovie: 30 s / 30 m

  16. Virus associates with proteasomes LMP2-GFP+mCherry-Vpr • Long-term association can be observed Virus for 90 minNo drugmovie: 30 s / 30 m

  17. Virus associates with proteasomes • Long-term association can be observed with a decrease in virus signal Virus for 90 minNo drugmovie: 30 s / 30 m

  18. Virus associates with proteasomes • Long-term association can be observed with a decrease in virus signal Virus for 90 minNo drugmovie: 30 s / 30 m LMP2-GFP+mCherry-Vpr

  19. Virus associates with proteasomes • Long-term association can be observed with a decrease in virus signal Virus for 90 minNo drugmovie: 30 s / 30 m LMP2-GFP+mCherry-Vpr

  20. Virus associates with proteasomes 9 minutes 14 minutes 19 minutes 24 minutes 29 minutes LMP2-GFP mCherry-Vpr 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.

  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. HA (TRIM)Rpn10mCherry-Vpr HA (TRIM)Rpt5mCherry-Vpr Ongoing Studies • Live cell imaging of proteasomes and rhTRIM5α • Is ubiquitin recruited before proteasomes? • Examining subunit composition of recruited proteasomes Tai and Schuman 2008

  23. ubiquitin Proteasome Capsid 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 rhTRIM5α

  24. Acknowledgments • Thomas J. Hope Laboratory • LMP2-GFP provided by J. Neefjes • Cells expressing HA-tagged rhTRIM5αprovided by J. Sodroski • Supported by T32 AI060523 and 5 R01 AI047770 Shannon Allen Ayanna Flegler Meegan Anderson Dan Gallo Ann Carias Amy Hulme Gianguido Cianci Ann Koons Minh Dinh Mike McRaven Doug Dylla Zia Okocha Carey Eppes Katharina Rothwangl Alison Erekson Christopher Rold Kelly Farhbach Eric Spongberg Anna Figueiredo Shetha Shukair Z Kelley

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