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

Cindy Danielson Thomas Hope Laboratory, Northwestern University

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rhTRIM5 α restricts HIV-1

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

rhTRIM5 α restricts HIV-1

• • • Early post-entry block Interacts with capsid core Results in failure to complete reverse transcription Wu 2006 Sawyer 2005

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rhTRIM5 α restricts HIV-1

rhTRIM5α restriction acts prior to reverse transcription Proteasome inhibition can rescue reverse transcription while maintaining block to infection Reverse Transcription Infection

rhT5



.HeLa

HeLa Mock HIV HIV.MG HIV.Nev

HIV HIV.MG

Wu 2006

Mechanism of Restriction

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

Consequence of ubiquitination

ubiquitin ubiquitin ubiquitin ubiquitin ubiquitin ubiquitin rhTRIM5 α ?

Capsid ?

Proteasomal degradation

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Characterizing ubiquitination

Polyubiquitination is dynamic in rhTRIM5α cytoplasmic bodies

+MG132

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Characterizing ubiquitination

rhTRIM5α-ΔK cytoplasmic bodies contain polyubiquitinated proteins 120 100 80 60 40 20 0

CONJUGATED POLYUBIQUITIN in cytoplasmic bodies

No drug MG132 Wild-type rhTRIM5α rhTRIM5α-ΔK

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Characterizing ubiquitination

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

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

No treatment

• Predominantly diffuse in nucleus

MG132

• Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with proteasome inhibition

Virus

• Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with virus

45 40 35 30 25 20 15 10 5 0

Proteasome localization

Proteasome relocalization to cytoplasmic bodies Mock MG132 Virus

• • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies Does virus interact with proteasomes?

Virus associates with proteasomes

• Long-term association can be observed Virus for 90 min No drug movie: 30 s / 30 m

Virus associates with proteasomes

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

Virus associates with proteasomes

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

Virus associates with proteasomes

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

Virus associates with proteasomes

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

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.

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 20 10 0 60 50 40 30 Virus Virus+MG132

Stable Stable+Dim Escape Diffuse

HA (TRIM) Rpn10 mCherry-Vpr HA (TRIM) Rpt5 mCherry-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

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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 rhTRIM5 α Capsid

Proteasome

Acknowledgments

• • • • Thomas J. Hope Laboratory Shannon Allen Meegan Anderson Ann Carias Gianguido Cianci Minh Dinh Doug Dylla Carey Eppes Alison Erekson Kelly Farhbach Anna Figueiredo Z Kelley Ayanna Flegler Dan Gallo Amy Hulme Ann Koons Mike McRaven Zia Okocha Katharina Rothwangl Christopher Rold Eric Spongberg Shetha Shukair LMP2-GFP provided by J. Neefjes Cells expressing HA tagged rhTRIM5α provided by J. Sodroski Supported by T32 AI060523 and 5 R01 AI047770