Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon

Abstract

The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1–72aa) is sufficient for viral transcript elongation and second exon (73–101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-κB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

“…9G). These data suggested that both Gag alone and Gag with accessory proteins could activate F-actin polymerization in T cells, as suggested in previous studies (44,45,66). Altogether, our data showed that Gag expression in Jurkat T cells activated Rac1 and increased the intracellular F-actin content.…”

“…The literature has reported an effect of the HIV-1 Tat and Nef accessory proteins on several Rac1-derived signaling pathways (44,45,66). We thus decided to study if the effect of the Rac1-Wave2-IRSp53-Arp3 signaling pathway on virus production observed here was mediated by HIV-1 accessory proteins or solely by the Gag viral protein.…”

“…F-actin measurement was performed by flow cytometry and immunofluorescence coupled to confocal microscopy image analysis, as previously described (66). The levels of polymerized actin were measured in 10 6 microporated cells (see above), in the absence of any stimuli.…”

“…9A). This effect might be due to the presence of the viral accessory proteins Tat and/or Nef, as it was reported previously that they could modulate Rac1 activity during the late steps of HIV-1 replication (66,73,74). Therefore, to determine the role of HIV-1 Gag alone in Rac1 activation, the same experiment was performed following pGag microporation in Jurkat T cells.…”

Protein Kinase Cθ Is a Specific Target for Inhibition of the HIV Type 1 Replication in CD4+ T Lymphocytes

“…DNA Affinity Immunoblotting Assay-The DNA affinity immunoblotting assay was performed as described previously (6) with minor modifications (42). Briefly, 100 g of nuclear protein extracts from Jurkat cells were incubated at 4°C for 30 min with 25 nM concentrations of a 5Ј-labeled biotin probe that contains the double B consensus motif located in the HIV-1 LTR promoter (5Ј-AGCTTACAAGGGACTTTCCGCTGGG-GACTTTCCAGGGA-3Ј) or with a probe that contains the estrogen receptor DNA element, a non-related NF-B gene (43).…”