The role of the ubiquitin-proteasome pathway in rhTRIM5α-mediated

restriction of HIV-1

Cindy DanielsonThomas Hope Laboratory, Northwestern University

rhTRIM5α restricts HIV-1• rhTRIM5α prevents infection of rhesus

macaques by HIV-1

Sawyer 2005

Towers 2005

rhTRIM5α restricts HIV-1• Early post-entry block• Interacts with capsid core• Results in failure to complete

reverse transcription

Sawyer 2005

Wu 2006

rhTRIM5α restricts HIV-1• rhTRIM5α restriction acts prior to reverse

transcription• Proteasome inhibition can rescue reverse

transcription while maintaining block to infection

Late

RT

/ Cel

l

0

5

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30

Mock HIV HIV.MG HIV.Nev%

GFP

+ ce

lls0

10

20

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HIV HIV.MG

HeLarhT5.HeLa

Wu 2006

Reverse Transcription Infection

Anderson 2006Campbell 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

Campbell 2008

GFP-Vpr HA (rhTRIM5α)

Consequence of ubiquitination

Proteasomal degradation

ubiquitin

ubiquitin

ubiquitin

rhTRIM5αCapsid

? ?

ubiquitin

ubiquitin

ubiquitin

• Polyubiquitination is dynamic in rhTRIM5α cytoplasmic bodies

Characterizing ubiquitination

+MG132

CONJUGATED POLYUBIQUITIN in cytoplasmic bodies

0

20

40

60

80

100

120

Wild-type rhTRIM5α rhTRIM5α-ΔK

No drugMG132

Characterizing ubiquitination• rhTRIM5α-ΔK cytoplasmic bodies contain

polyubiquitinated proteins

Nor

mal

ized

ubi

quiti

n/pu

ncta

te H

A

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

• Predominantly diffuse in nucleus

No treatment

MG132

• Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with proteasome inhibition

Virus

• Proteasomes relocalize to rhTRIM5α cytoplasmic bodies with virus

Proteasome relocalization to cytoplasmic bodies

05

1015202530354045

Mock MG132 Virus

• Proteasomes relocalize to rhTRIM5α cytoplasmic bodies

• Does virus interact with proteasomes?

Proteasome localization#

cyto

plas

mic

pro

teas

ome

punc

ta/c

ell

Virus associates with proteasomes

Virus for 90 minNo drugmovie: 30 s / 30 m

• Long-term association can be observed

Virus associates with proteasomes

Virus for 90 minNo drugmovie: 30 s / 30 m

• Long-term association can be observed

LMP2-GFP + mCherry-Vpr

Virus associates with proteasomes

Virus for 90 minNo drugmovie: 30 s / 30 m

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

Virus associates with proteasomes

Virus for 90 minNo drugmovie: 30 s / 30 m

LMP2-GFP + mCherry-Vpr

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

Virus associates with proteasomes

Virus for 90 minNo drugmovie: 30 s / 30 m

LMP2-GFP + mCherry-Vpr

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

Virus associates with proteasomes

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.

9 minutes 14 minutes 19 minutes 24 minutes 29 minutes

LMP2-GFP mCherry-Vpr

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

0

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Stable Stable+Dim Escape Diffuse

% o

f tot

al as

socia

tions

h

VirusVirus+MG132

Ongoing Studies• Live cell imaging of

proteasomes and rhTRIM5α

• Is ubiquitin recruited before proteasomes?

• Examining subunit composition of recruited proteasomes

HA (TRIM) Rpn10mCherry-Vpr

HA (TRIM) Rpt5mCherry-Vpr

Tai and Schuman 2008

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

CapsidrhTRIM5α

Proteasome

Acknowledgments• Thomas J. Hope Laboratory

• LMP2-GFP provided by J. Neefjes• Cells expressing HA-tagged rhTRIM5α

provided by J. Sodroski• Supported by T32 AI060523 and 5 R01 AI047770

Shannon Allen Ayanna FleglerMeegan Anderson Dan GalloAnn Carias Amy HulmeGianguido Cianci Ann KoonsMinh Dinh Mike McRavenDoug Dylla Zia OkochaCarey Eppes Katharina

RothwanglAlison Erekson Christopher RoldKelly Farhbach Eric SpongbergAnna Figueiredo Shetha ShukairZ Kelley