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.
Highlights
The human immunodeficiency virus type-1 (HIV-1)1 is the etiologic agent of the AIDS, a progressive and degenerative disease of the immune system [1]
The molecular mechanisms underlying the transcriptional function of Tat have been clarified by showing that Tat interacts with the p-TEFb complex, which includes the Cdk9 cyclin-dependent kinase physically associated to members of cyclin T such as T1, T2a, and T2b [12, 31, 32, 70]
We used the yeast two-hybrid system to identify Tatinteracting proteins and identified a cDNA clone encoding for E2F-4, a transcription factor that plays a key role in the regulation of the cell cycle progression [42,43,44,45]
Summary
The human immunodeficiency virus type-1 (HIV-1)1 is the etiologic agent of the AIDS, a progressive and degenerative disease of the immune system [1]. To verify the ability of wild-type E2F-4 to interact with HIV-1 Tat protein, the full-length E2F-4 cDNA coding for amino acids 2– 413 was cloned downstream to the GAL-4 activation domain in the pACT yeast expression vector.
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