Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription
<p>Abstract</p> <p>Background</p> <p>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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2005-02-01
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| Series: | Virology Journal |
| Online Access: | http://www.virologyj.com/content/2/1/17 |
| _version_ | 1819183167652757504 |
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| author | Harrich David Warby Tammra Endo-Munoz Liliana McMillan Nigel AJ |
| author_facet | Harrich David Warby Tammra Endo-Munoz Liliana McMillan Nigel AJ |
| author_sort | Harrich David |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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. <it>In vitro </it>phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type <it>tat </it>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 <it>in vivo </it>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.</p> <p>Conclusion</p> <p>These results support the hypothesis that phosphorylation of Tat may be important for its function in HIV-1 LTR transactivation.</p> |
| first_indexed | 2024-12-22T22:57:43Z |
| format | Article |
| id | doaj.art-3bd59eb9ec0f40f291047dec7fd821b2 |
| institution | Directory Open Access Journal |
| issn | 1743-422X |
| language | English |
| last_indexed | 2024-12-22T22:57:43Z |
| publishDate | 2005-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Virology Journal |
| spelling | doaj.art-3bd59eb9ec0f40f291047dec7fd821b22022-12-21T18:09:46ZengBMCVirology Journal1743-422X2005-02-01211710.1186/1743-422X-2-17Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcriptionHarrich DavidWarby TammraEndo-Munoz LilianaMcMillan Nigel AJ<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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. <it>In vitro </it>phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type <it>tat </it>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 <it>in vivo </it>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.</p> <p>Conclusion</p> <p>These results support the hypothesis that phosphorylation of Tat may be important for its function in HIV-1 LTR transactivation.</p>http://www.virologyj.com/content/2/1/17 |
| spellingShingle | Harrich David Warby Tammra Endo-Munoz Liliana McMillan Nigel AJ Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription Virology Journal |
| title | Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription |
| title_full | Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription |
| title_fullStr | Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription |
| title_full_unstemmed | Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription |
| title_short | Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription |
| title_sort | phosphorylation of hiv tat by pkr increases interaction with tar rna and enhances transcription |
| url | http://www.virologyj.com/content/2/1/17 |
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