Mitogen-Activated Protein Kinases Regulate LSF Occupancy at the Human Immunodeficiency Virus Type 1 Promoter

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by cycles of virus production and reinfection within activated CD4+ T lymphocytes. The efficiency of HIV-1 replication diminishes as cells enter the resting state, but this lentivirus can replicate and persist within resting cell populations (55, 56). Although suppressive antiretroviral therapy is often clinically successful, eradication of HIV infection is currently unattainable due to the persistent, quiescent infection of resting CD4 memory T cells (31). Global activation of T cells appears to be an untenable therapeutic strategy to disrupt latency of HIV (45), and recently induction of HIV expression without full activation of T cells has been proposed as an alternative approach. Evidence suggests that signaling pathways exist that may reactivate virus expression in primary cells (2, 22, 24), and these pathways may serve as targets for future therapies. Consequently, it is important to understand in detail the mechanisms that regulate the transition from latent to productive infection. Two human transcription factors, YY1 and LSF, play a role in the establishment or maintenance of HIV latency via changes in local chromatin architecture surrounding the HIV promoter (8, 9, 35, 53). LSF (late simian virus 40 [SV40] transcription factor) is a member of the LSF/CP2 subfamily of transcription factors (41, 47). LSF was initially identified as a factor that binds to and stimulates transcription from the viral SV40 major late promoter (20). LSF is involved in the regulation of several genes, including those for interleukin 4 (IL-4) (4), human α-globin (25), thymidylate synthase (33), and the HIV long terminal repeat (LTR) (19, 35). YY1 is a highly conserved GLI-Kruppel transcription factor that can function as activator, repressor, or initiator of transcription depending on the context of its binding site within a promoter and its interaction with other proteins. Of note, YY1 can downregulate transcription through recruitment of histone deacetylases (34, 51, 52). Recent data suggest that this mechanism of regulation of HIV expression is relevant in latent infection of resting CD4+ T cells. Krishnan and Zeichner have found that YY1 expression is upregulated in latently infected cells prior to activation and expression of HIV (21). We have shown that histone deacetylase (HDAC) inhibition potently induces expression of latent HIV from the resting CD4+ T cells of HIV-infected patients (54). LSF and YY1 form a complex at the HIV LTR that recruits HDAC1 and represses LTR transcription. Coexpression of YY1 and LSF synergistically inhibits LTR-directed gene expression and HIV virus production. Three binding motifs have been identified within the HIV promoter at a region from −10 to +27 surrounding the transcriptional start site designated the repressor complex sequence (RCS). LSF binds the RCS, but YY1 is required to repress LTR transcription. In contrast, YY1 does not form a specific protein complex with the RCS, and an LSF mutant unable to bind DNA blocks repression of the LTR by YY1. In turn, YY1 mutants unable to bind HDAC lack the ability to repress the LTR (8, 28, 35). Mitogen-activated protein kinase (MAPK) pathways regulate entry into the cell cycle (48). The three central MAPKs, Erk, Jnk, and p38, are phosphorylated on both threonine and tyrosine residues by dual specificity by MAP kinase kinases (MEKs), which in turn are activated by serine/threonine phosphorylation by MAP kinase kinase kinase. Mammalian Erk1 and Erk2 and their upstream activators MEK1 and MEK2 are stimulated by signaling of growth and differentiation factors, such as epidermal growth factor, platelet-derived growth factor, and nerve growth factor, through receptor tyrosine kinases, heterotrimeric G protein-coupled receptors, or cytokine receptors. The mammalian Jnks and p38 are implicated in responses to cellular stress, inflammation, and apoptosis (16). In response to extracellular stimuli, MAPKs are activated in the cytoplasm and can rapidly be transported into the nucleus. Once activated, MAPKs induce phosphorylation of transcription factors present in the cytoplasm or nucleus. The phosphorylation of transcription factors by MAPK can change their affinity for DNA or change their affinity for other transcription factors regulating the expression of specific genes (40). After mitogenic stimulation of human peripheral blood mononuclear cells (PBMCs), LSF is rapidly phosphorylated by Erk1/Erk2. In primary T cells, Erk signaling is correlated with a rapid and transient increase in the ability of LSF to bind DNA (30, 43, 44). However, Erk phosphorylation in vitro is not sufficient to increase its binding to the LSF site. We sought to further understand the LSF binding to the HIV LTR and the role of phosphorylation in regulating this event.