The role of the ubiquitin proteasome pathway in rhtrim5 mediated restriction of hiv 1
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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 rhtrim5 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 restricts hiv 1
rhTRIM5 rhTRIM5α-mediated restriction of HIV-1α restricts HIV-1

  • rhTRIM5α prevents infection of rhesus macaques by HIV-1

Sawyer 2005

Towers 2005


Rhtrim5 restricts hiv 11
rhTRIM5 rhTRIM5α-mediated restriction of HIV-1α restricts HIV-1

  • Early post-entry block

  • Interacts with capsid core

  • Results in failure to complete reverse transcription

Wu 2006

Sawyer 2005


Rhtrim5 restricts hiv 12

Late RT / Cell rhTRIM5α-mediated restriction of HIV-1

% 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


Mechanism of restriction

GFP-Vpr rhTRIM5α-mediated restriction of HIV-1HA (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


Consequence of ubiquitination

ubiquitin rhTRIM5α-mediated restriction of HIV-1

ubiquitin

ubiquitin

ubiquitin

ubiquitin

ubiquitin

Capsid

Consequence of ubiquitination

rhTRIM5α

?

?

Proteasomal degradation


Characterizing ubiquitination
Characterizing ubiquitination rhTRIM5α-mediated restriction of HIV-1

  • Polyubiquitination is dynamic in rhTRIM5α cytoplasmic bodies

+MG132


Characterizing ubiquitination1
Characterizing ubiquitination rhTRIM5α-mediated restriction of HIV-1

  • rhTRIM5α-ΔK cytoplasmic bodies contain polyubiquitinated proteins

Normalized ubiquitin/punctate HA


Characterizing ubiquitination2
Characterizing ubiquitination rhTRIM5α-mediated restriction of HIV-1

  • 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

Tai and Schuman 2008 rhTRIM5α-mediated restriction of HIV-1

What is the role of the proteasome?

  • Imaging approach using a fluorescent proteasome construct

    • LMP2-GFP is a βsubunit of active proteasomes


No treatment rhTRIM5α-mediated restriction of HIV-1

  • Predominantly diffuse in nucleus


MG132 rhTRIM5α-mediated restriction of HIV-1

  • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with proteasome inhibition


Virus rhTRIM5α-mediated restriction of HIV-1

  • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with virus


Proteasome localization
Proteasome localization rhTRIM5α-mediated restriction of HIV-1

  • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies

  • Does virus interact with proteasomes?

# cytoplasmic proteasome puncta/cell


Virus associates with proteasomes
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

  • Long-term association can be observed

Virus for 90 minNo drugmovie: 30 s / 30 m


Virus associates with proteasomes1
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

LMP2-GFP+mCherry-Vpr

  • Long-term association can be observed

Virus for 90 minNo drugmovie: 30 s / 30 m


Virus associates with proteasomes2
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

  • Long-term association can be observed with a decrease in virus signal

Virus for 90 minNo drugmovie: 30 s / 30 m


Virus associates with proteasomes3
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

  • 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


Virus associates with proteasomes4
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

  • 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


Virus associates with proteasomes5
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

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 proteasomes6
Virus associates with proteasomes rhTRIM5α-mediated restriction of HIV-1

  • Proteasomes and virus associate in live cells

    • Stable association

    • Stable association with decrease in virus signal

    • Escape

    • Proteasome accumulation becomes more diffuse


Ongoing studies

HA (TRIM) rhTRIM5α-mediated restriction of HIV-1Rpn10mCherry-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


Conclusions

ubiquitin rhTRIM5α-mediated restriction of HIV-1

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


Acknowledgments
Acknowledgments rhTRIM5α-mediated restriction of HIV-1

  • 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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