Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription

Endo-Munoz, Liliana, Warby, Tammra, Harrich, David and McMillan, Nigel A. J. (2005) Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription. Virology Journal, 2 17.1-17.13. doi:10.1186/1743-422X-2-17


Author Endo-Munoz, Liliana
Warby, Tammra
Harrich, David
McMillan, Nigel A. J.
Title Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription
Journal name Virology Journal   Check publisher's open access policy
ISSN 1743-422X
Publication date 2005-02
Sub-type Article (original research)
DOI 10.1186/1743-422X-2-17
Open Access Status DOI
Volume 2
Start page 17.1
End page 17.13
Total pages 13
Editor Robert F. Garry
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2005
Language eng
Subject C1
321015 Oncology and Carcinogenesis
730108 Cancer and related disorders
Formatted abstract
Background:
The interferon (IFN)-induced, dsRNA-dependent serine/threonine protein kinase, PKR, plays a key regulatory role in the IFN-mediated anti-viral response by blocking translation in the infected cell by phosphorylating the alpha subunit of elongation factor 2 (eIF2). The human immunodeficiency virus type 1 (HIV-1) evades the anti-viral IFN response through the binding of one of its major transcriptional regulatory proteins, Tat, to PKR. HIV-1 Tat acts as a substrate homologue for the enzyme, competing with eIF2α, and inhibiting the translational block. It has been shown that during the interaction with PKR, Tat becomes phosphorylated at three residues: serine 62, threonine 64 and serine 68. We have investigated the effect of this phosphorylation on the function of Tat in viral transcription. HIV-1 Tat activates transcription elongation by first binding to TAR RNA, a stem-loop structure found at the 5' end of all viral transcripts. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR.

Results:
We have investigated the effect of phosphorylation on Tat-mediated transactivation. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. In vitro phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type tat gene or mutants of the gene with alanine substitutions at one, two, or all three of the serine/threonine PKR phosphorylation sites, showed that these were subject to different levels of phosphorylation by PKR and displayed distinct kinetic behaviour. These results also suggested a cooperative role for the phosphorylation of S68 in conjunction with S62 and T64. We examined the effect of phosphorylation on Tat-mediated transactivation of the HIV-1 LTR in vivo with a series of analogous mammalian expression constructs. Co-transfection experiments showed a gradual reduction in transactivation as the number of mutated phosphorylation sites increased, and a 4-fold decrease in LTR transactivation with the Tat triple mutant that could not be phosphorylated by PKR. Furthermore, the transfection data also suggested that the presence of S68 is necessary for optimal Tat-mediated transactivation.

Conclusion:
These results support the hypothesis that phosphorylation of Tat may be important for its function in HIV-1 LTR transactivation.
© 2005 Endo-Munoz et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keyword Alanine
Protein kinase R
Regulator protein
Transactivator protein
Virus protein
Amino acid substitution
Enzyme phosphorylation
Gene construct
Gene expression system
Genetic transfection
Human immunodeficiency virus
HIV
Protein phosphorylation
Protein protein interaction
Protein RNA binding
RNA structure
Transcription regulation
Virus transcription
Wild type
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

 
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Created: Wed, 15 Aug 2007, 07:33:42 EST