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Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.

Ammosova T, Berro R, Jerebtsova M, Jackson A, Charles S, Klase Z, Southerland W, Gordeuk VR, Kashanchi F, Nekhai S - Retrovirology (2006)

Bottom Line: CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat.Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells.Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Sickle Cell Disease, Howard University College of Medicine, Washington, DC 20059, USA. tammosova@mail.ru

ABSTRACT

Background: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

Results: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease.

Conclusion: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

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A, Mutations of S16 and S46 of Tat reduce its ability to induce viral production. HLM1 is a HeLa derived cells containing one copy of integrated HIV-1 proviral genome with a Tat-defective mutation. Various Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A) expression vectors were used for HLM-1 transfections. Cells were cultured in complete media in absence of G418 for 14 days. The supernatants were collected at Day 0, 1, 2, 7 and 14, and analyzed for p24 by ELISA assay. B, Mutations of S16 and S46 of Tat inhibit Tat phosphorylation during one round of viral replication. HLM1 cells were transfected with vectors expressing Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A). At 48 hours post transfection the cells were labeled with (32P)-orthophosphate for 2 hours with 1 μM okadaic acid. Flag-Tat was immunoprecipitated from whole cell extracts with anti-Flag antibodies and resolved by 15% Tris-Tricine SDS-PAGE. The gel was dried and exposed to Phosphor Imager screen. Lane 1, Wt Tat. Lane 2, Tat S16A. Lane 3, Tat S46A. Lane 4, Tat S16,46A. Lane 5, mock-transfected cells. The picture is an autoradiogram.
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Figure 10: A, Mutations of S16 and S46 of Tat reduce its ability to induce viral production. HLM1 is a HeLa derived cells containing one copy of integrated HIV-1 proviral genome with a Tat-defective mutation. Various Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A) expression vectors were used for HLM-1 transfections. Cells were cultured in complete media in absence of G418 for 14 days. The supernatants were collected at Day 0, 1, 2, 7 and 14, and analyzed for p24 by ELISA assay. B, Mutations of S16 and S46 of Tat inhibit Tat phosphorylation during one round of viral replication. HLM1 cells were transfected with vectors expressing Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A). At 48 hours post transfection the cells were labeled with (32P)-orthophosphate for 2 hours with 1 μM okadaic acid. Flag-Tat was immunoprecipitated from whole cell extracts with anti-Flag antibodies and resolved by 15% Tris-Tricine SDS-PAGE. The gel was dried and exposed to Phosphor Imager screen. Lane 1, Wt Tat. Lane 2, Tat S16A. Lane 3, Tat S46A. Lane 4, Tat S16,46A. Lane 5, mock-transfected cells. The picture is an autoradiogram.

Mentions: We determined whether mutations of Tat S16A and/or S46A have an effect on the ability of Tat to induce HIV-1 transcription from an integrated HIV-1 provirus. We used HLM-1 cells (AIDS Research and Reference Reagent Program) that were derived from HeLa-CD4+ cells containing an integrated copy of HIV-1 proviral genome with a Tat-defective mutation (termination linker at the first AUG). HLM-1 cells are negative for virus particle production, but can be induced to express high levels of infectious HIV-1 after transfection with Tat. We transfected the HLM-1 cells with wild type or mutant Tat vectors and tested supernatants for the presence of HIV-1 particles using p24 gag antigen ELISA at day 0, day 1, day 2, day 7 and day 14 posttransfection. Neither S16A nor S46A mutants of Tat efficiently induced HIV-1 viral production (Fig. 10A). The double S16, 46A mutant also had a reduced activity (Fig. 10A). To phosphorylate Tat during virus replication, we pulsed HLM-1 cells transfected with WT and mutant Tat with (32P) orthophosphate and also treated the cells with okadaic acid. Tat was immunoprecipitated with anti-Flag antibodies, resolved on 15% SDS PAGE and its phosphorylation was detected by PhosphoImager. While WT Tat was phosphorylated, the mutants were not phosphorylated efficiently (Fig. 10B). These data indicate that the S16A and S46A mutations of Tat interfere with the ability of Tat to activate integrated HIV-1 provirus, and prevent Tat phosphorylation during one round of viral replication.


Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription.

Ammosova T, Berro R, Jerebtsova M, Jackson A, Charles S, Klase Z, Southerland W, Gordeuk VR, Kashanchi F, Nekhai S - Retrovirology (2006)

A, Mutations of S16 and S46 of Tat reduce its ability to induce viral production. HLM1 is a HeLa derived cells containing one copy of integrated HIV-1 proviral genome with a Tat-defective mutation. Various Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A) expression vectors were used for HLM-1 transfections. Cells were cultured in complete media in absence of G418 for 14 days. The supernatants were collected at Day 0, 1, 2, 7 and 14, and analyzed for p24 by ELISA assay. B, Mutations of S16 and S46 of Tat inhibit Tat phosphorylation during one round of viral replication. HLM1 cells were transfected with vectors expressing Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A). At 48 hours post transfection the cells were labeled with (32P)-orthophosphate for 2 hours with 1 μM okadaic acid. Flag-Tat was immunoprecipitated from whole cell extracts with anti-Flag antibodies and resolved by 15% Tris-Tricine SDS-PAGE. The gel was dried and exposed to Phosphor Imager screen. Lane 1, Wt Tat. Lane 2, Tat S16A. Lane 3, Tat S46A. Lane 4, Tat S16,46A. Lane 5, mock-transfected cells. The picture is an autoradiogram.
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Related In: Results  -  Collection

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Figure 10: A, Mutations of S16 and S46 of Tat reduce its ability to induce viral production. HLM1 is a HeLa derived cells containing one copy of integrated HIV-1 proviral genome with a Tat-defective mutation. Various Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A) expression vectors were used for HLM-1 transfections. Cells were cultured in complete media in absence of G418 for 14 days. The supernatants were collected at Day 0, 1, 2, 7 and 14, and analyzed for p24 by ELISA assay. B, Mutations of S16 and S46 of Tat inhibit Tat phosphorylation during one round of viral replication. HLM1 cells were transfected with vectors expressing Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A). At 48 hours post transfection the cells were labeled with (32P)-orthophosphate for 2 hours with 1 μM okadaic acid. Flag-Tat was immunoprecipitated from whole cell extracts with anti-Flag antibodies and resolved by 15% Tris-Tricine SDS-PAGE. The gel was dried and exposed to Phosphor Imager screen. Lane 1, Wt Tat. Lane 2, Tat S16A. Lane 3, Tat S46A. Lane 4, Tat S16,46A. Lane 5, mock-transfected cells. The picture is an autoradiogram.
Mentions: We determined whether mutations of Tat S16A and/or S46A have an effect on the ability of Tat to induce HIV-1 transcription from an integrated HIV-1 provirus. We used HLM-1 cells (AIDS Research and Reference Reagent Program) that were derived from HeLa-CD4+ cells containing an integrated copy of HIV-1 proviral genome with a Tat-defective mutation (termination linker at the first AUG). HLM-1 cells are negative for virus particle production, but can be induced to express high levels of infectious HIV-1 after transfection with Tat. We transfected the HLM-1 cells with wild type or mutant Tat vectors and tested supernatants for the presence of HIV-1 particles using p24 gag antigen ELISA at day 0, day 1, day 2, day 7 and day 14 posttransfection. Neither S16A nor S46A mutants of Tat efficiently induced HIV-1 viral production (Fig. 10A). The double S16, 46A mutant also had a reduced activity (Fig. 10A). To phosphorylate Tat during virus replication, we pulsed HLM-1 cells transfected with WT and mutant Tat with (32P) orthophosphate and also treated the cells with okadaic acid. Tat was immunoprecipitated with anti-Flag antibodies, resolved on 15% SDS PAGE and its phosphorylation was detected by PhosphoImager. While WT Tat was phosphorylated, the mutants were not phosphorylated efficiently (Fig. 10B). These data indicate that the S16A and S46A mutations of Tat interfere with the ability of Tat to activate integrated HIV-1 provirus, and prevent Tat phosphorylation during one round of viral replication.

Bottom Line: CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat.Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells.Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Sickle Cell Disease, Howard University College of Medicine, Washington, DC 20059, USA. tammosova@mail.ru

ABSTRACT

Background: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

Results: We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser16 and Ser46 residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with 32P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser16 and Ser46 residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser16 and Ser46 residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S16QP(K/R)19 and S46YGR49 sequences of Tat showed that Ser16 and Ser46 and R49 residues are highly conserved whereas mutation of the (K/R)19 residue correlated with non-progression of HIV-1 disease.

Conclusion: Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.

Show MeSH
Related in: MedlinePlus