Structural and functional insights into IκB-α/HIV-1 Tat interaction.

Protein-protein interactions play fundamental roles in physiological and pathological biological processes. The characterization of the structural determinants of protein-protein recognition represents an important step for the development of molecular entities able to modulate these interactions. We have recently found that IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) blocks the HIV-1 expression and replication in a NF-κB-independent manner by directly binding to the virus-encoded Tat transactivator. Here, we report the evaluation of the entity of binding of IκB-α to Tat through in vitro Surface Plasmon Resonance assay. Moreover, by designing and characterizing a set of peptides of the C-terminus region of IκB-α, we show that the peptide corresponding to the IκB-α sequence 262-287 was able to bind to Tat with high affinity (300 nM). The characterization of a number of IκB-α-based peptides also provided insights into their intrinsic folding properties. These findings have been corroborated by mutagenesis studies on the full-length IκB-α, which unveil that different IκB-α residues are involved in NF-κB or Tat recognition.

IkappaB-alpha represses the transcriptional activity of the HIV-1 Tat transactivator by promoting its nuclear export.

The long terminal repeat of human immunodeficiency virus, type 1 (HIV-1) contains an NF-kappaB enhancer and is potently inhibited by IkappaB-alphaS32/36A, a proteolysis-resistant inhibitor of NF-kappaB transacting factors. The evidence that NF-kappaB is dispensable for HIV-1 expression raises the question of whether IkappaB-alpha represses the HIV-1 transcription by mechanisms distinct from NF-kappaB inhibition. Here, we report that IkappaB-alpha negatively regulates the HIV-1 expression and replication in an NF-kappaB-independent manner by directly binding to Tat, which results in the nuclear export and cytoplasmic sequestration of the viral transactivator. The sequence of IkappaB-alpha required for Tat inhibition spans from amino acids 72 to 287 and includes the nuclear localization signal, the carboxyl-terminal nuclear export signal, and the binding site for the arginine-rich domain of Tat. This novel mechanism of cross-talk between Tat and IkappaB-alpha provides further insights into the mechanisms of HIV-1 regulation and could assist in the development of novel strategies for AIDS therapy.

Inhibition of HIV-1 replication in primary human monocytes by the IkappaB-alphaS32/36A repressor of NF-kappaB.

BACKGROUND: The identification of the molecular mechanisms of human immunodeficiency virus type 1, HIV-1, transcriptional regulation is required to develop novel inhibitors of viral replication. NF-kappaB transacting factors strongly enhance the HIV/SIV expression in both epithelial and lymphoid cells. Controversial results have been reported on the requirement of NF-kappaB factors in distinct cell reservoirs, such as CD4-positive T lymphocytes and monocytes. We have previously shown that IkappaB-alphaS32/36A, a proteolysis-resistant inhibitor of NF-kappaB, potently inhibits the growth of HIV-1 and SIVmac239 in cell cultures and in the SIV macaque model of AIDS. To further extend these observations, we have generated NL(AD8)IkappaB-alphaS32/36A, a macrophage-tropic HIV-1 recombinant strain endowed to express IkappaB-alphaS32/36A. RESULTS: In this work, we show that infection with NL(AD8)IkappaB-alphaS32/36A down-regulated the NF-kappaB DNA binding activity in cells. NL(AD8)IkappaB-alphaS32/36A was also highly attenuated for replication in cultures of human primary monocytes. CONCLUSIONS: These results point to a major requirement of NF-kappaB activation for the optimal replication of HIV-1 in monocytes and suggest that agents which interfere with NF-kappaB activity could counteract HIV-1 infection of monocytes-macrophages in vivo.

High attenuation and immunogenicity of a simian immunodeficiency virus expressing a proteolysis-resistant inhibitor of NF-kappaB.

NF-kappaB/IkappaB proteins play a major role in the transcriptional regulation of human immunodeficiency virus, type-1 (HIV-1). In the case of simian immunodeficiency virus (SIV) the cellular factors required for the viral transcriptional activation and replication in vivo remain undefined. Here, we demonstrate that the p50/p65 NF-kappaB transcription factors enhanced the Tat-mediated transcriptional activation of SIVmac239. In addition, IkappaB-alpha S32/36A, a proteolysis-resistant inhibitor of NF-kappaB, strongly inhibited the Tat-mediated transactivation of SIVmac239. Based on this evidence, we have generated a self-regulatory virus by endowing the genome of SIV-mac239 with IkappaB-alpha S32/36A; the resulting virus, SIVIkappaB-alpha S32/36A, was nef-deleted and expressed the NF-kappaB inhibitor. We show that SIVIkappaB-alpha S32/36A was highly and stably attenuated both in cell cultures and in vivo in rhesus macaque as compared with a nef-deleted control virus. Moreover, the high attenuation was associated with a robust immune response as measured by SIV-specific antibody production, tetramer, and intracellular IFN-gamma staining of SIV gag-specific T cells. These results underscore the crucial role of NF-kappaB/IkappaB proteins in the regulation of SIV replication both in cell cultures and in monkeys. Thus, inhibitors of NF-kappaB could efficiently counteract the SIV/HIV replication in vivo and may assist in developing novel approaches for AIDS vaccine and therapy.

Physical and functional interaction of HIV-1 Tat with E2F-4, a transcriptional regulator of mammalian cell cycle.

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.