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rhTRIM5α

The role of the ubiquitin-proteasome pathway in rhTRIM5α-mediated restriction of HIV-1 Cindy Danielson Thomas Hope Laboratory, Northwestern University. rhTRIM5α. rhTRIM5α blocks infection of rhesus macaques by HIV-1. Sawyer 2005. IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA.

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rhTRIM5α

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  1. The role of the ubiquitin-proteasome pathwayin rhTRIM5α-mediated restriction of HIV-1Cindy DanielsonThomas Hope Laboratory, Northwestern University

  2. rhTRIM5α rhTRIM5α blocks infection of rhesus macaques by HIV-1 Sawyer 2005 IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Towers 2005

  3. rhTRIM5α rhTRIM5α blocks infection of rhesus macaques by HIV-1 -Early post-entry block -Interacts with capsid core -Results in failure to complete reverse transcription IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Wu 2006 Sawyer 2005

  4. Late RT / Cell % GFP+ cells rhT5.HeLa HeLa Reverse Transcription Infection Mock HIV HIV.MG HIV.Nev HIV HIV.MG rhTRIM5α rhTRIM5α blocks infection of rhesus macaques by HIV-1 -rhTRIM5α restriction acts prior to reverse transcription -Proteasome inhibition can rescue reverse transcription while maintaining block to infection IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Wu 2006

  5. GFP-Vpr HA (rhTRIM5α) Campbell 2008 Anderson 2006 Campbell 2008 rhTRIM5α Mechanism of restriction -Recognition of capsid core by rhTRIM5α -Proteasome-sensitive block of reverse transcription -Proteasome inhibitors allow visualization of intermediate stage -Ubiquitin localizes with rhTRIM5α IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  6. ubiquitin ubiquitin ubiquitin ubiquitin ubiquitin ubiquitin rhTRIM5α Capsid What is the role of the proteasome? Does ubiquitin in cytoplasmic bodies lead to degradation? ? ? IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Proteasomal degradation

  7. What is the role of the proteasome? Imaging approach using a fluorescent proteasome construct -LMP2-GFP is a β subunit of active proteasomes IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Tai and Schuman 2008

  8. Proteasome localization No treatment -Proteasomes predominantly diffuse in nucleus IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  9. Proteasome localization MG132 -Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with MG132 IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  10. Proteasome localization Virus -Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with virus IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  11. Proteasome localization Proteasomes are recruited to rhTRIM5α cytoplasmic bodies -Proteasomes relocalize to rhTRIM5α cytoplasmic bodies -Does virus interact with proteasomes? IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  12. Virus associates with proteasomes Long-termassociation can be observed IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus for 90 minNo drugmovie: 30 s / 30 m

  13. Virus associates with proteasomes Long-termassociation can be observed LMP2-GFPmCherry-Vpr IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus for 90 minNo drugmovie: 30 s / 30 m

  14. Virus associates with proteasomes Long-termassociation with a decrease in virus signal LMP2-GFPmCherry-Vpr IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus for 90 minNo drugmovie: 30 s / 30 m

  15. Virus associates with proteasomes Long-termassociation with a decrease in virus signal IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus for 90 minNo drugmovie: 30 s / 30 m LMP2-GFP+mCherry-Vpr

  16. Virus associates with proteasomes Long-termassociation with a decrease in virus signal IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus for 90 minNo drugmovie: 30 s / 30 m LMP2-GFP+mCherry-Vpr

  17. Virus associates with proteasomes Long-termassociation with a decrease in virus signal 9 minutes 14 minutes 19 minutes 24 minutes 29 minutes IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA 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.

  18. Virus associates with proteasomes Types of associations and effect of proteasome inhibition -Stable association-Stable association with decrease in virus signal-Escape of virus-Associated proteasome accumulation becomes more diffuse IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  19. Ongoing studies HA (TRIM)Rpn10mCherry-Vpr HA (TRIM)Rpt5mCherry-Vpr -Live cell imaging of proteasomes and rhTRIM5α-Is ubiquitin recruited before proteasomes? -Examining subunit composition of recruited proteasomes IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  20. Conclusions -Proteasome localization is dynamic -Proteasomes localize to rhTRIM5α cytoplasmic bodies -Relocalization induced by virus or by proteasome inhibition-Virus associates with proteasomes in living cells -Several types of association can be observed -Effect of proteasome inhibition-Highlights the importance of cellular factors in establishing a productive infection IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

  21. Acknowledgments -Thomas J. Hope LaboratoryShannon Allen Meegan Anderson Ann CariasGianguido Cianci Minh Dinh Doug DyllaCarey Eppes Alison Erekson Kelly FarhbachAnna Figueiredo Ayanna Flegler Dan GalloAmy Hulme Ann Koons Mike McRavenZia Okocha Katharina Rothwangl Christopher RoldEric Spongberg Shetha Shukair Z Kelley -LMP2-GFP provided by J. Neefjes -Cells expressing HA-tagged rhTRIM5α provided by J. Sodroski -Supported by National Institutes of Health T32 AI060523 and 5 R01 AI047770 IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA

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