Abstract
Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.
Publication types
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Research Support, N.I.H., Intramural
MeSH terms
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Blotting, Western
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Cell Cycle Proteins
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Cell Nucleus / genetics
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Cell Nucleus / metabolism
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Chromatin Immunoprecipitation
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Cyclin T / genetics
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Cyclin T / metabolism
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Flow Cytometry
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Gene Products, tax / genetics
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Gene Products, tax / metabolism*
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HTLV-I Infections / genetics
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HTLV-I Infections / metabolism
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HTLV-I Infections / virology
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HeLa Cells / metabolism
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HeLa Cells / virology
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Human T-lymphotropic virus 1 / pathogenicity
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Humans
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Molecular Weight
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Positive Transcriptional Elongation Factor B / genetics
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Positive Transcriptional Elongation Factor B / metabolism*
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Protein Binding
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RNA, Messenger / genetics
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RNA, Small Interfering / pharmacology
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism*
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Reverse Transcriptase Polymerase Chain Reaction
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Ribonucleoproteins, Small Nuclear / genetics
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Ribonucleoproteins, Small Nuclear / metabolism*
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Terminal Repeat Sequences / genetics
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcription, Genetic
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Virus Replication
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gag Gene Products, Human Immunodeficiency Virus / genetics
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gag Gene Products, Human Immunodeficiency Virus / metabolism
Substances
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BRD4 protein, human
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Cell Cycle Proteins
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Cyclin T
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Gene Products, tax
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HEXIM1 protein, human
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Nuclear Proteins
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RNA, Messenger
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RNA, Small Interfering
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RNA-Binding Proteins
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Ribonucleoproteins, Small Nuclear
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Transcription Factors
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gag Gene Products, Human Immunodeficiency Virus
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p19 protein, Human T-lymphotropic virus 1
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tax protein, Human T-lymphotrophic virus 1
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Positive Transcriptional Elongation Factor B