The role of the ubiquitin proteasome pathway in rhtrim5 mediated restriction of hiv 1
This presentation is the property of its rightful owner.
Sponsored Links
1 / 24

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


  • 90 Views
  • Uploaded on
  • Presentation posted in: General

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.

Download Presentation

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

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


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


The role of the ubiquitin proteasome pathway in rhtrim5 mediated restriction of hiv 1

No treatment

  • Predominantly diffuse in nucleus


The role of the ubiquitin proteasome pathway in rhtrim5 mediated restriction of hiv 1

MG132

  • Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with proteasome inhibition


The role of the ubiquitin proteasome pathway in rhtrim5 mediated restriction of hiv 1

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 AllenAyanna Flegler

Meegan AndersonDan Gallo

Ann CariasAmy Hulme

Gianguido CianciAnn Koons

Minh DinhMike McRaven

Doug DyllaZia Okocha

Carey EppesKatharina Rothwangl

Alison EreksonChristopher Rold

Kelly FarhbachEric Spongberg

Anna FigueiredoShetha Shukair

Z Kelley


  • Login