Limits...
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.

Show MeSH

Related in: MedlinePlus

Mutations of Ser16 and Ser46 of Tat reduce its transactivation potential. A, COS-7 cells were transfected with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, the cells were lysed. Tat was immunoprecipitated from the lysates with rabbit polyclonal antibodies, resolved by 15% Tris-Tricine SDS-PAGE and immunoblotted with monoclonal anti-Tat antibodies. B, HeLa cells were transfected with the HIV-1 LTR-LacZ expression vector alone (not shown here) and in combination with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, cells were lysed and analyzed for β-galactosidase activity with ONPG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1636661&req=5

Figure 9: Mutations of Ser16 and Ser46 of Tat reduce its transactivation potential. A, COS-7 cells were transfected with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, the cells were lysed. Tat was immunoprecipitated from the lysates with rabbit polyclonal antibodies, resolved by 15% Tris-Tricine SDS-PAGE and immunoblotted with monoclonal anti-Tat antibodies. B, HeLa cells were transfected with the HIV-1 LTR-LacZ expression vector alone (not shown here) and in combination with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, cells were lysed and analyzed for β-galactosidase activity with ONPG.

Mentions: We next investigated the functional relevance of Tat's S16 and S46 residues in HIV-1 transcription. We generated mutants of Tat in which either or both Ser residues were substituted by Ala. To ensure expression of the mutants, COS-7 cells were transfected with WT and mutant Tat-expressing vectors and cellular lysates were analyzed on 15% SDS Tris-Tricine PAGE followed by Western blot with anti-Tat antibodies. As shown in Fig. 9A, all Tat mutants were expressed, with the level of expression of Tat mutants higher than the WT Tat. The higher expression level of non-tagged Tat mutants was a reproducible effect and was not a consequence of the difference in the amount of transfected DNA. The effect of Tat mutations on the ability of Tat to activate HIV-1 LTR promoter was analyzed in HeLa cells co-transfected with Tat-expression vectors and HIV-1 LTR-LacZ reporter plasmid (Fig. 9B). Non-mutated Tat (WT) increased the level of transcription by 400-fold (Fig 9B). HIV-1 transcription induction by the Tat S16A mutant was approximately 75% that of WT Tat (Fig. 9B), while transactivation by the Tat S46A mutant was about 2 times lower than with the WT Tat (Fig. 9B) and induction by the double S16, 46A Tat mutant was 3-times lower than that of WT Tat (Fig. 9B). Thus, mutation of either Ser16 or Ser46 of Tat interferes with the level of Tat-transactivation and mutation of both residues has an additive effect.


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)

Mutations of Ser16 and Ser46 of Tat reduce its transactivation potential. A, COS-7 cells were transfected with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, the cells were lysed. Tat was immunoprecipitated from the lysates with rabbit polyclonal antibodies, resolved by 15% Tris-Tricine SDS-PAGE and immunoblotted with monoclonal anti-Tat antibodies. B, HeLa cells were transfected with the HIV-1 LTR-LacZ expression vector alone (not shown here) and in combination with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, cells were lysed and analyzed for β-galactosidase activity with ONPG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636661&req=5

Figure 9: Mutations of Ser16 and Ser46 of Tat reduce its transactivation potential. A, COS-7 cells were transfected with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, the cells were lysed. Tat was immunoprecipitated from the lysates with rabbit polyclonal antibodies, resolved by 15% Tris-Tricine SDS-PAGE and immunoblotted with monoclonal anti-Tat antibodies. B, HeLa cells were transfected with the HIV-1 LTR-LacZ expression vector alone (not shown here) and in combination with WT Tat, Tat S16A, Tat S46A or Tat S16,46A expression vectors. At 48 hours posttransfection, cells were lysed and analyzed for β-galactosidase activity with ONPG.
Mentions: We next investigated the functional relevance of Tat's S16 and S46 residues in HIV-1 transcription. We generated mutants of Tat in which either or both Ser residues were substituted by Ala. To ensure expression of the mutants, COS-7 cells were transfected with WT and mutant Tat-expressing vectors and cellular lysates were analyzed on 15% SDS Tris-Tricine PAGE followed by Western blot with anti-Tat antibodies. As shown in Fig. 9A, all Tat mutants were expressed, with the level of expression of Tat mutants higher than the WT Tat. The higher expression level of non-tagged Tat mutants was a reproducible effect and was not a consequence of the difference in the amount of transfected DNA. The effect of Tat mutations on the ability of Tat to activate HIV-1 LTR promoter was analyzed in HeLa cells co-transfected with Tat-expression vectors and HIV-1 LTR-LacZ reporter plasmid (Fig. 9B). Non-mutated Tat (WT) increased the level of transcription by 400-fold (Fig 9B). HIV-1 transcription induction by the Tat S16A mutant was approximately 75% that of WT Tat (Fig. 9B), while transactivation by the Tat S46A mutant was about 2 times lower than with the WT Tat (Fig. 9B) and induction by the double S16, 46A Tat mutant was 3-times lower than that of WT Tat (Fig. 9B). Thus, mutation of either Ser16 or Ser46 of Tat interferes with the level of Tat-transactivation and mutation of both residues has an additive effect.

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