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rhTRIM5α restricts HIV-1 Early post-entry block Interacts with capsid core Results in failure to complete reverse transcription Sawyer 2005 Wu 2006
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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
10
15
20
25
30
Mock HIV HIV.MG HIV.Nev%
GFP
+ ce
lls0
10
20
30
40
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
10
20
30
40
50
60
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