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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
rhTRIM5α restricts HIV-1
  • rhTRIM5α prevents infection of rhesus macaques by HIV-1

Sawyer 2005

Towers 2005

rhtrim5 restricts hiv 11
rhTRIM5α 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

% 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 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
consequence of ubiquitination

ubiquitin

ubiquitin

ubiquitin

ubiquitin

ubiquitin

ubiquitin

Capsid

Consequence of ubiquitination

rhTRIM5α

?

?

Proteasomal degradation

characterizing ubiquitination
Characterizing ubiquitination
  • Polyubiquitination is dynamic in rhTRIM5α cytoplasmic bodies

+MG132

characterizing ubiquitination1
Characterizing ubiquitination
  • rhTRIM5α-ΔK cytoplasmic bodies contain polyubiquitinated proteins

Normalized ubiquitin/punctate HA

characterizing ubiquitination2
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

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
slide11

No treatment

  • Predominantly diffuse in nucleus
slide12

MG132

  • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with proteasome inhibition
slide13

Virus

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

# cytoplasmic proteasome puncta/cell

virus associates with proteasomes
Virus associates with proteasomes
  • Long-term association can be observed

Virus for 90 minNo drugmovie: 30 s / 30 m

virus associates with proteasomes1
Virus associates with proteasomes

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
  • 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
  • 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
  • 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

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
  • 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)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

conclusions

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

acknowledgments
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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