TRBP directs specific HIV1 mRNAs to polysomes for translation

1
TRBP directs specific HIV-1 mRNAsto polysomes
for translation

• Shahan Campbell
• Molecular Virology Laboratory
• Dept. of Microbiology and Immunology

2
Viral dsRNA stimulates PKR activity

Antiviral response
PKR
dsRNA
3
Viral dsRNA stimulates PKR activity

Antiviral response
PKR
McMillan et al 1995 Virology 213 413 Brand et al
1997 JBC 2728388
Tat
dsRNA
4
TRBP inhibits PKR activity

Antiviral response
PKR
Benkirane et al 1997 EMBO J 16(3) 611 Park et al
1994 PNAS 91 4713
McMillan et al 1995 Virology 213 413 Brand et al
1997 JBC 2728388
TRBP
Tat
dsRNA
5
TRBP inhibits PKR activity

Antiviral response
PKR
Benkirane et al 1997 EMBO J 16(3) 611 Park et al
1994 PNAS 91 4713
McMillan et al 1995 Virology 213 413 Brand et al
1997 JBC 2728388
TRBP
Tat
dsRNA
Dicer
Haase et al 2005 EMBO Reports. 6(10)961 Chendrima
da et al 2005 Nature 436 740
6
TRBP inhibits PKR activity

Antiviral response
PKR
Benkirane et al 1997 EMBO J 16(3) 611 Park et al
1994 PNAS 91 4713
McMillan et al 1995 Virology 213 413 Brand et al
1997 JBC 2728388
TRBP
Tat
dsRNA
Dicer
Bennasser et al 2005 Immunity 22 607
Haase et al 2005 EMBO Reports. 6(10)961 Chendrima
da et al 2005 Nature 436 740
7
HYPOTHESISRNA structures in HIV mRNAcontrol
the state of translation
Non-permissive U251MG Astrocytoma cells
Low TRBP
Tat

Viral mRNA Tat and Rev proteins Gag, Pol Env
proteins Virion release

TRBP
/-
/-
Gorry et al. J Virol. 1999 73(1)352 Ong et al. J
Virol. 2005 79(20)12763

• TAR and RRE interact with cell molecules to
  facilitate active translation

8
HYPOTHESISRNA structures in HIV mRNAcontrol
the state of translation
Non-permissive U251MG Astrocytoma cells
Low TRBP
Tat

Viral mRNA Tat and Rev proteins Gag, Pol Env
proteins Virion release

TRBP
/-
/-
Gorry et al. J Virol. 1999 73(1)352 Ong et al. J
Virol. 2005 79(20)12763

• TAR and RRE interact with cell molecules to
  facilitate active translation

9
env mRNA is more responsive to TRBP than nef mRNA
RRE only (CMV-env mRNA)
TAR only (LTR-nef mRNA)
TAR and RRE (LTR-env mRNA)
10
env mRNA is more responsive to TRBP than nef mRNA
env mRNA
nef mRNA
-
-


TRBP plasmid
TRBP plasmid
Env
Nef
B-actin
B-actin
TRBP
TRBP
Christensen H. et al. Poster WEPEA026
11
Gradient isolation of polysomes
12
Characteristic regions of gradient
Top
Bottom
Density (g/mL)
Untreated
A260nm
Puromycin 0.5mg/mL
gapdh mRNA (total)
gapdh mRNA distribution
F r a c t I o n s
13
Characteristic regions of gradient
Top
Bottom
Density (g/mL)
Untreated
A260nm
Puromycin 0.5mg/mL
gapdh mRNA (total)
gapdh mRNA distribution
F r a c t I o n s
14
Characteristic regions of gradient
Top
Bottom
Density (g/mL)
Untreated
A260nm
Puromycin 0.5mg/mL
translating polysomes
gapdh mRNA (total)
gapdh mRNA distribution
F r a c t I o n s
15
Characteristic regions of gradient
Top
Bottom
Density (g/mL)
Untreated
A260nm
Puromycin 0.5mg/mL
translating polysomes
L5 (ribosome marker)
Dcp1a (p-body marker)
F r a c t I o n s
16
TRBP effect on nef and env mRNA distribution
nef mRNA
env mRNA
With TRBP
Without TRBP
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TRBP effect on nef and env mRNA distribution
nef mRNA
env mRNA
With TRBP
Without TRBP
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TRBP effect on nef and env mRNA distribution
nef mRNA
env mRNA
With TRBP
Without TRBP
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PKR distribution and activation state
nef mRNA
PKR
without TRBP
p-PKR
endogenous TRBP
polysomes
PKR
with TRBP
p-PKR
TRBP
1 2 3 4 5 6 7 8 9
10 11 12
p-PKR phosphorylated PKR
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PKR distribution and activation state
env mRNA
PKR
without TRBP
p-PKR
endogenous TRBP
polysomes
PKR
with TRBP
p-PKR
TRBP
1 2 3 4 5 6 7 8 9 10
11 12
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Conclusions

• Overexpression of TRBP protein leads to migration
  of TRBP with polysomes
• TRBP promotes the accumulation of HIV-1 mRNAs in
  the translation-active polysome cell compartment
• TRBP preferentially increases the translation of
  env mRNAs that accumulate on polysomes compared
  to nef mRNAs
• TRBP inhibits local PKR phosphorylation - induced
  by env mRNAs on polysomes

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Acknowledgements

• University of Melbourne
• Francine Fangsi Ke
• Helen Christensen
• Jane Howard
• Marina Alexander
• Sean Chung
• Adam Johnson
• Damian Purcell
• McGill University
• Anne Gatignol

University of Colorado Jens Lykke-Andersen
Oregon Health and Science University Hua
Lu Burnet Institute Alison Greenway Funding Uni
versity of Melbourne National Health and Medical
Research Council - Australia
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