The presence of HIV-1 Tat protein second exon delays fas protein-mediated apoptosis in CD4+ T lymphocytes: a potential mechanism for persistent viral production.

HIV-1 replication is efficiently controlled by the regulator protein Tat (101 amino acids) and codified by two exons, although the first exon (1-72 amino acids) is sufficient for this process. Tat can be released to the extracellular medium, acting as a soluble pro-apoptotic factor in neighboring cells. However, HIV-1-infected CD4(+) T lymphocytes show a higher resistance to apoptosis. We observed that the intracellular expression of Tat delayed FasL-mediated apoptosis in both peripheral blood lymphocytes and Jurkat cells, as it is an essential pathway to control T cell homeostasis during immune activation. Jurkat-Tat cells showed impairment in the activation of caspase-8, deficient release of mitochondrial cytochrome c, and delayed activation of both caspase-9 and -3. This protection was due to a profound deregulation of proteins that stabilized the mitochondrial membrane integrity, such as heat shock proteins, prohibitin, or nucleophosmin, as well as to the up-regulation of NF-κB-dependent anti-apoptotic proteins, such as BCL2, c-FLIPS, XIAP, and C-IAP2. These effects were observed in Jurkat expressing full-length Tat (Jurkat-Tat101) but not in Jurkat expressing the first exon of Tat (Jurkat-Tat72), proving that the second exon, and particularly the NF-κB-related motif ESKKKVE, was necessary for Tat-mediated protection against FasL apoptosis. Accordingly, the protection exerted by Tat was independent of its function as a regulator of both viral transcription and elongation. Moreover, these data proved that HIV-1 could have developed strategies to delay FasL-mediated apoptosis in infected CD4(+) T lymphocytes through the expression of Tat, thus favoring the persistent replication of HIV-1 in infected T cells.

Protein kinase Ctheta is a specific target for inhibition of the HIV type 1 replication in CD4+ T lymphocytes.

Integration of HIV-1 genome in CD4(+) T cells produces latent reservoirs with long half-life that impedes the eradication of the infection. Control of viral replication is essential to reduce the size of latent reservoirs, mainly during primary infection when HIV-1 infects CD4(+) T cells massively. The addition of immunosuppressive agents to highly active antiretroviral therapy during primary infection would suppress HIV-1 replication by limiting T cell activation, but these agents show potential risk for causing lymphoproliferative disorders. Selective inhibition of PKC, crucial for T cell function, would limit T cell activation and HIV-1 replication without causing general immunosuppression due to PKC being mostly expressed in T cells. Accordingly, the effect of rottlerin, a dose-dependent PKC inhibitor, on HIV-1 replication was analyzed in T cells. Rottlerin was able to reduce HIV-1 replication more than 20-fold in MT-2 (IC(50) = 5.2 µM) and Jurkat (IC(50) = 2.2 µM) cells and more than 4-fold in peripheral blood lymphocytes (IC(50) = 4.4 µM). Selective inhibition of PKC, but not PKCδ or -ζ, was observed at <6.0 µM, decreasing the phosphorylation at residue Thr(538) on the kinase catalytic domain activation loop and avoiding PKC translocation to the lipid rafts. Consequently, the main effector at the end of PKC pathway, NF-κB, was repressed. Rottlerin also caused a significant inhibition of HIV-1 integration. Recently, several specific PKC inhibitors have been designed for the treatment of autoimmune diseases. Using these inhibitors in combination with highly active antiretroviral therapy during primary infection could be helpful to avoid massive viral infection and replication from infected CD4(+) T cells, reducing the reservoir size at early stages of the infection.

Changes in the cellular microRNA profile by the intracellular expression of HIV-1 Tat regulator: A potential mechanism for resistance to apoptosis and impaired proliferation in HIV-1 infected CD4+ T cells.

HIV-1 induces changes in the miRNA expression profile of infected CD4+ T cells that could improve viral replication. HIV-1 regulator Tat modifies the cellular gene expression and has been appointed as an RNA silencing suppressor. Tat is a 101-residue protein codified by two exons that regulates the elongation of viral transcripts. The first exon of Tat (amino acids 1-72) forms the transcriptionally active protein Tat72, but the presence of the second exon (amino acids 73-101) results in a more competent regulatory protein (Tat101) with additional functions. Intracellular, full-length Tat101 induces functional and morphological changes in CD4+ T cells that contribute to HIV-1 pathogenesis such as delay in T-cell proliferation and protection against FasL-mediated apoptosis. But the precise mechanism by which Tat produces these changes remains unknown. We analyzed how the stable expression of intracellular Tat101 and Tat72 modified the miRNA expression profile in Jurkat cells and if this correlated with changes in apoptotic pathways and cell cycle observed in Tat-expressing cells. Specifically, the enhanced expression of hsa-miR-21 and hsa-miR-222 in Jurkat-Tat101 cells was associated with the reduced expression of target mRNAs encoding proteins related to apoptosis and cell cycle such as PTEN, PDCD4 and CDKN1B. We developed Jurkat cells with stable expression of hsa-miR-21 or hsa-miR-222 and observed a similar pattern to Jurkat-Tat101 in resistance to FasL-mediated apoptosis, cell cycle arrest in G2/M and altered cell morphology. Consequently, upregulation of hsa-miR-21 and hsa-miR-222 by Tat may contribute to protect against apoptosis and to anergy observed in HIV-infected CD4+ T cells.

Mechanisms of RNA interference in the HIV-1-host cell interplay.

The RNA interference pathway is a mechanism to regulate gene expression that acts in mammalian antiviral immunity as a complement of interferon- and cytokine-based innate immunity. RNA interference has been proposed as an ancient mechanism against viruses since several components of this system show an effect against viral replication. In fact, protein effectors of this pathway, as well as synthesized microRNA, act against HIV-1, exerting a partial control over HIV-1 latency and replication. Conversely, HIV-1 may counteract this antiviral cell response through two major lines of attack: first, its main regulator Tat suppresses the cellular RNA interference pathway; and second, the virus synthesizes viral microRNA that alter specific cellular functions to enhance HIV-1 replication. As a result of this complex interaction, the microRNA profile in an HIV-1-infected cell is deeply modified. One key application of all this knowledge is the development of pharmacological treatment since microRNA expression can be manipulated and artificial small interference RNA can be delivered into the infected cell to inhibit viral replication. This strategy, combined with current antiretroviral therapy, could be valuable in controlling HIV-1 replication in infected cells.

Dual role of host cell factors in HIV-1 replication: restriction and enhancement of the viral cycle.

Once HIV-1 enters the target cell, the first goal in the viral cycle is to integrate into the cellular chromosomes. The irreversible integration as a provirus allows HIV-1 to persist in the infected cell in a quiescent or latent stage that leads to viral escape from immune response and current antiviral treatment. HIV-1 replication is absolutely dependent on different cellular and viral factors that initiate viral expression, acting at the long terminal repeat of the integrated provirus. Accordingly, HIV-1 induces changes in the cellular environment to make possible an efficient replication and production of viral progeny. One main instigator of HIV-1 replication is the viral regulator Tat, which is absolutely required for efficient transcription and elongation of viral transcripts. For this purpose, Tat recruits several cellular proteins to make the chromatin structure accessible for the transcription machinery, to acquire the posttranslational modifications essential for its function, and to produce efficient viral replication. However, the host cell has also several antiviral mechanisms that may act at different steps of the viral cycle to thwart HIV-1 replication. To level the match, HIV-1 encodes accessory proteins, such as Vif and Vpu, which play important roles in HIV-1 pathogenesis by counteracting cellular antiviral factors. The increasing knowledge of viral protein interactions with host cell factors will be essential for the discovery of new targets that could be used to design new therapeutic strategies.