The role of the ubiquitinproteasome pathway in rh
The role of the ubiquitin-proteasome pathway in rh. TRIM 5α-mediated restriction of HIV-1 Cindy Danielson Thomas Hope Laboratory, Northwestern University
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA rh. TRIM 5α blocks infection of rhesus macaques by HIV-1 Sawyer 2005 Towers 2005
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA rh. TRIM 5α blocks infection of rhesus macaques by HIV-1 -Early post-entry block -Interacts with capsid core -Results in failure to complete reverse transcription Wu 2006 Sawyer 2005
rh. TRIM 5α blocks infection of rhesus macaques by HIV-1 -rh. TRIM 5α restriction acts prior to reverse transcription -Proteasome inhibition can rescue reverse transcription while maintaining block to infection Reverse Transcription Infection % GFP+ cells Late RT / Cell IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA rh. TRIM 5α Mock HIV. MG HIV. Nev rh. T 5. He. La HIV. MG Wu 2006
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA rh. TRIM 5α Mechanism of restriction -Recognition of capsid core by rh. TRIM 5α -Proteasome-sensitive block of reverse transcription -Proteasome inhibitors allow visualization of intermediate stage -Ubiquitin localizes with rh. TRIM 5α GFP-Vpr HA (rh. TRIM 5α) Anderson 2006 Campbell 2008
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA What is the role of the proteasome? Does ubiquitin in cytoplasmic bodies lead to degradation? ubiquitin ? ? ubiquitin Proteasomal degradation rh. TRIM 5α Capsid
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA What is the role of the proteasome? Imaging approach using a fluorescent proteasome construct -LMP 2 -GFP is a β subunit of active proteasomes Tai and Schuman 2008
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Proteasome localization No treatment -Proteasomes predominantly diffuse in nucleus
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Proteasome localization MG 132 -Proteasomes relocalize to rh. TRIM 5α cytoplasmic bodies with MG 132
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Proteasome localization Virus -Proteasomes relocalize to rh. TRIM 5α cytoplasmic bodies with virus
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Proteasome localization Proteasomes are recruited to rh. TRIM 5α cytoplasmic bodies -Proteasomes relocalize to rh. TRIM 5α cytoplasmic bodies -Does virus interact with proteasomes?
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Long-term association can be observed Virus for 90 min No drug movie: 30 s / 30 m
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Long-term association can be observed LMP 2 -GFP m. Cherry-Vpr Virus for 90 min No drug movie: 30 s / 30 m
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Long-term association with a decrease in virus signal LMP 2 -GFP m. Cherry-Vpr Virus for 90 min No drug movie: 30 s / 30 m
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Long-term association with a decrease in virus signal Virus for 90 min No drug movie: 30 s / 30 m LMP 2 -GFP + m. Cherry-Vpr
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Long-term association with a decrease in virus signal Virus for 90 min No drug movie: 30 s / 30 m LMP 2 -GFP + m. Cherry-Vpr
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Long-term association with a decrease in virus signal 9 minutes 14 minutes 19 minutes 24 minutes 29 minutes LMP 2 -GFP m. Cherry-Vpr Time course of a live cell experiment in which cells stably expressing HA-tagged rh. TRIM 5α were transfected with LMP 2 -GFP and infected with m. Cherry-Vpr labeled virus. Times are indicated from the start of imaging. m. Cherry-Vpr can be seen associated with an accumulation of LMP 2 -GFP for the duration of the movie (30 minutes) and the fluorescent signal of the virus appears to decrease during this association.
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Virus associates with proteasomes Types of associations and effect of proteasome inhibition -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 Ongoing studies -Live cell imaging of proteasomes and rh. TRIM 5α HA (TRIM) Rpn 10 m. Cherry-Vpr -Is ubiquitin recruited before proteasomes? -Examining subunit composition of recruited proteasomes HA (TRIM) Rpt 5 m. Cherry-Vpr
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Conclusions -Proteasome localization is dynamic -Proteasomes localize to rh. TRIM 5α 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 Acknowledgments -Thomas J. Hope Laboratory Shannon Allen Meegan Anderson Ann Carias Gianguido Cianci Minh Doug Dylla Carey Eppes Alison Erekson Kelly Farhbach Anna Figueiredo Ayanna Flegler Dan Gallo Amy Hulme Ann Koons Mike Mc. Raven Zia Okocha Katharina Rothwangl Christopher Rold Eric Spongberg Shetha Shukair Z Kelley -LMP 2 -GFP provided by J. Neefjes -Cells expressing HA-tagged rh. TRIM 5α provided by J. Sodroski -Supported by National Institutes of Health T 32 AI 060523 and 5 R 01 AI 047770
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