title: Physical and functional interaction of HIV-1 Tat with E2F-4, a transcriptional regulator of mammalian cell cycle.
abstract: Tat protein of the human immunodeficiency virus type-1 (HIV-1) plays a critical role in the regulation of viral transcription and replication. In addition, Tat regulates the expression of a variety of cellular genes and could account for AIDS-associated diseases including Kaposi's Sarcoma and non-Hodgkin's lymphoma by interfering with cellular processes such as proliferation, differentiation, and apoptosis. The molecular mechanisms underlying the pleiotropic activities of Tat may include the generation of functional heterodimers of Tat with cellular proteins. By screening a human B-lymphoblastoid cDNA library in the yeast two-hybrid system, we identified E2F-4, a member of E2F family of transcription factors, as a Tat-binding protein. The interaction between Tat and E2F-4 was confirmed by GST pull-down experiments performed with cellular extracts as well as with in vitro translated E2F-4. The physical association of Tat and E2F-4 was confirmed by in vivo binding experiments where Tat.E2F-4 heterodimers were recovered from Jurkat cells by immunoprecipitation and immunoblotting. By using plasmids expressing mutant forms of Tat and E2F-4, the domains involved in Tat.E2F-4 interaction were identified as the regions encompassing amino acids 1-49 of Tat and amino acids 1-184 of E2F-4. Tat x E2F-4 complexes were shown to bind to E2F cis-regions with increased efficiency compared with E2F-4 alone and to mediate the activity of E2F-dependent promoters including HIV-1 long terminal repeat and cyclin A. The data point to Tat as an adaptor protein that recruits cellular factors such as E2F-4 to exert its multiple biological activities.
authors.affiliation: Department of Clinical and Experimental Medicine, Medical School, University of Catanzaro, 88100 Catanzaro, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Ambrosino
authors.forename: Camillo
authors.initials: C
authors.lastname: Palmieri
authors.forename: Antimina
authors.initials: A
authors.lastname: Puca
authors.forename: Francesca
authors.initials: F
authors.lastname: Trimboli
authors.forename: Marco
authors.initials: M
authors.lastname: Schiavone
authors.forename: Francesco
authors.initials: F
authors.lastname: Olimpico
authors.forename: Maria R
authors.initials: MR
authors.lastname: Ruocco
authors.forename: Francesca
authors.initials: F
authors.lastname: di Leva
authors.forename: Mario
authors.initials: M
authors.lastname: Toriello
authors.forename: Ileana
authors.initials: I
authors.lastname: Quinto
authors.forename: Salvatore
authors.initials: S
authors.lastname: Venuta
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Scala
mesh_terms: D001402:B-Lymphocytes; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D019281:Dimerization; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase