title: A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription.
abstract: BACKGROUND
  Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II.
  
  
  RESULTS
  To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus.
  
  
  CONCLUSIONS
  This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription.
authors.forename: Alona
authors.initials: A
authors.lastname: Kuzmina
authors.forename: Nina
authors.initials: N
authors.lastname: Verstraete
authors.forename: Sigal
authors.initials: S
authors.lastname: Galker
authors.forename: Maayan
authors.initials: M
authors.lastname: Maatook
authors.forename: Olivier
authors.initials: O
authors.lastname: Bensaude
authors.affiliation: The Shraga Segal Department of Microbiology, Immunology and Genetics Faculty of Health Sciences, Ben-Gurion University of the Negev, P,O, Box 653, Beer-Sheva 84105, ISRAEL. rantaube@bgu.ac.il.
authors.forename: Ran
authors.initials: R
authors.lastname: Taube
mesh_terms: D016475:3T3 Cells; D000818:Animals; D056906:Cyclin T / Q000737:chemistry / Q000378:metabolism*; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D057809:HEK293 Cells; D006678:HIV / Q000235:genetics*; D006801:Humans; D051379:Mice; D017354:Point Mutation; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000821:virology; D014157:Transcription Factors; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000502:physiology
chemical_list: C471348:AFF4 protein, human; C503684:CCNT1 protein, human; D056906:Cyclin T; C476123:HEXIM1 protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9