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HIV encapsidates viral genomic RNA and APOBEC3G in mRNA
processing bodies
Renato S Aguiar
Federal University of Rio de Janeiro (UFRJ) - Brazil
University of California, San Francisco (UCSF) - USA
Virus RNA
Translation Packaging
P bodies
Today’s Talk
Virus assembly
Genomic RNA incorporation
mRNA processing bodies (P bodies) role
Functions of unspliced viral RNA
CAP AAAA incorporation
translationassembly
Cell cytoplasm
Virus RNA
translationtranslation
How these two different functions are coordinated?
P-bodies components
APOBEC3G
Staufen
Cellular proteins that are important for HIV replication
Active translation mRNA storage
P-bodies components
APOBEC3G
Staufen
Active translation mRNA storage
P bodies mRNA storage and degradation
• cell development
• neuron differentiation
• RNA viruses
Newbury et al., EMBO Reports 2006
CAP AAAA
Transcription
Translation
Storage
Stress Granules
P bodies
Starvation
Viral infection
Stress
CAP A
Degradation
mRNA decay
Cellular RNA metabolism
exossomes
P Bodies Knockdown of P bodies structural proteins
GW182
Localize to P bodies and have been shown to function inRNA silencing.
RCK/p54
ATP-dependent RNA helicase. Rearrange RNA structures or dissociate RNA-protein complexes.
Disruption of P bodiesTZM-bl Cells – 72h postransfection
Immunofluorescense
GW182
++
+
actin
GW182
mockcontrol siRNA
GW182 siRNA
++
+
actin
control siRNA
RCK/p54 DAPI merge RCK/p54 DAPI merge
GW182 siRNA
mockcontrol siRNA
RCK/p54 siRNA
++
+RCK/p54
actin
eIF4E-T DAPI merge
control siRNA
RCK/p54 siRNA
GW182 knockdown RCK/p54 knockdown
WB
siRNA transfectionsi c- RNA, si GW182, si RCK
TZM-bl
60h pos knockdown
HIV (NL43)
Virus budded in supernatants
5h infectionwash
48h pos-infection
Fresh TZM-bl cellsIndicator cells
5h infection
24h pos infection
Cell extraction luciferase assay
Infectivity of virus produced from P-bodies
depleted cells
P Bodies role in HIV replication
luciferaseLTR
tattat
TZM-bl cell lysate
Infectivity of HIV virus produced from P bodies depleted cells
Producer cellsReduction of HIV infectivity in virus
produced from P bodies depleted cells
0
20
40
60
80
100
Infe
ctiv
ity (
%)
mock control GW182 RCKsiRNA:
siRNA:ControlGW182
RCK
+ ++
+GW182
actin
p55
RCK
1 2 3 4 p24
Does P-bodies interfere in HIV release or genomic RNA incorporation?
0
50
100
150
200
250
300
mock control GW182 RCK
p24
leve
ls (
pg/m
l)
siRNA:
Virus Purification
Sucrose cushion
Virus
RT-PCR (cDNA)
Real Time PCR
gag region
Virus release – p24
genomic RNA incorporation
mock control GW182 RCK
rela
tive
RN
A le
vels
0
0.2
0.4
0.6
0.8
1.0
1.2Cells Virus
siRNA:
P bodies are important for HIV
RNA incorporation
These structures can help in HIV assembly. Storage HIV genomic
RNA avoiding degradation.
Swanson & Malim, Traffic, 2006
NEXT STEPS Binding of virus RNA and P bodies components
RNA – Immunoprecipitation (similar to CHIP)
HIV RNA
Tag- Protein
clearing
Ab specific to Tag-protein
Beads binds AbsWash-precipitation
process
Release of RNA
cDNA
qPCRcgag primers
HIV RNA
P bodies Tag proteins
293T
transfection
PCR cycles
Gag f:Ago2 h:eIF4E h:RCK GW182.GFP
input
IP:IgG
IP:specific
30 35 40 30 35 40 30 35 40 30 35 40 30 35 40
RNA
RNA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
LTR LTRGag Pol Env
HIV:
b.
+ + + + +HIV
f:Ago2
h:RCK
h:eIF4E
GW182.GFP
++
++
WB-lysate
p55
1 2 3 4 5
a.
1144 1429
Positive control
P bodies proteins binds HIV genomic RNA
• Ago2
• eIF4E
• RCK
siRNA:control
RCKsiRNA:
Viral RNA accumulates in P Bodies
Removal of P bodies disperses HIV RNA
HIV RNA DAPI merge
Ago2.GFP HIV RNA DAPI merge
FISH
a.b. c.
Incorporation of transient transfected
Ago2 into viral particles
Endogenous Ago2 incorporation into viral
particles is dependent of P bodies
HIV released from primary cells
incorporate Ago2
293T cells293T cells
macrophage
lymphocyte
A P body component is incorporated into new viral particles Ago2 was the only P body component to be incorporated at
detectable levels into VLPs
Identification of Host Proteins Required for HIV Infection
Abraham L. Brass, et al. 2008
Argonaute
microRNA (miRNA) and short interfering RNA
(siRNA) function
Ago2.GFP αGag DAPI merge
GW182.GFP αGag mergeDAPI
GAG ALSO ACCUMULATES IN P BODIES
p55
Gag + Ago2
Flag
f:Ago2input
p55
RNAse A: +- -IgG
p55
GFP
+- -IgG
GW.GFP
p55
p55
h:RCK
p55
HA
+- -IgG HA
+- -IgG
h:eIF4E
p55
p55 IP
Structural virus protein (Gag) interacts with P bodies and this interaction is dependent of RNA
Co-immunoprecipitation (Gag + P bodies proteins)
Gag + GW Gag + RCK Gag + eIF4E
virion
lysate
virion
lysate
1 2 3 4
+ + + + ++ + ++
++
v:A3G
v:A3G
p24
p55
GW182
RCK
Actin
HIVVifv:A3G
siRNA: controlGW182
RCK
APOBEC3G incorporation into viral particles requires P bodies
Inhibit HIV Vif replication
KewalRamani & Coffin, Science 2003
Summary
These results are innovative and demonstrate that P bodies are required for HIV replication.
Our results suggest that these compartments are important for virus assembly and genomic RNA incorporation into new HIV particles.
Genomic RNA and Gag proteins co-localized and co-precipitated with components of P bodies, which were subsequently incorporated into new viral particles.
Depletion of P bodies also dispersed HIV genomic RNA in the cytoplasm.
Finally, these foci were also required for the incorporation of the host restriction factor APOBEC3G into progeny virions.
This observation suggests that interfering with P bodies could decrease HIV replication.
Significance
• Matija Peterlin (UCSF-USA)
• Xavier Contreras (UCSF-USA)
• Amilcar Tanuri (UFRJ)
• Luciana Costa (UFRJ)
• Ana Luiza Valadão (UFRJ)
Acknowledgments
Funding
UFRJ-Brazil UCSF-USA
contact: [email protected]
Infe
ctiv
ity (
%)
020406080
100120140160180200
control RCKsiRNA:
48 h
controlIn
fect
ivity
(%
)0
20
40
60
80
100
siRNA: RCK
siRNA:
eIF4E-T
48 h 60 h
control
RCK
120 h
siRNA:
RCK 0 24 48 60 120 h:
Actin
1 2 3 4 5
Time after tranfection
RCK siRNA
DELAYED DISAPPEARANCE OF P BODIES AFTER KNOCKDOWN OF RCK
60 h
