CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.
Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150.
Suñé
C
C
Department of Molecular Cancer Biology, Levine Science Research Center, Duke University Medical Center, Durham, North Carolina 27710, USA.
Hayashi
T
T
Liu
Y
Y
Lane
W S
WS
Young
R A
RA
Garcia-Blanco
M A
MA
D000409:Alanine; D000595:Amino Acid Sequence; D002457:Cell Extracts; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D015696:Gene Products, tat / Q000502:physiology; D005973:Glutamine; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*; D035602:Transcriptional Elongation Factors; D054322:tat Gene Products, Human Immunodeficiency Virus
D002457:Cell Extracts; D003067:Coenzymes; D015696:Gene Products, tat; D009687:Nuclear Proteins; C108440:TCERG1 protein, human; D015534:Trans-Activators; D035602:Transcriptional Elongation Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D005973:Glutamine; D012319:RNA Polymerase II; D000409:Alanine