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FOXP3 is a necessary transcription factor for the development and function of CD4+ regulatory T-cells (Tregs). The role of Tregs in HIV-1 infection remains unclear. Here, we show that expression of FOXP3 in primary human CD4 T-cells significantly inhibits HIV-1 infection. Since FOXP3 inhibits NFAT activity, and NFAT proteins contribute to HIV-1 transcription, we explore a transcriptional repressive function of HIV-1 LTR by FOXP3. Over-expression of FOXP3 in primary CD4 T-cells inhibits wild-type HIV-1 LTR reporter activity, and truncation mutants demonstrate that repression of the LTR by FOXP3 requires the dual proximal NF kappaB/NFAT binding sites. Interestingly, FOXP3 decreases binding of NFAT2 to the HIV-1 LTR in vivo. Furthermore, FOXP3 does not inhibit infection of HIV-1 NL4-3 which is mutated to disrupt transcription factor binding at either proximal NFAT or NF kappaB binding sites. These data suggest that resistance of Tregs to HIV-1 infection is due to inhibition of HIV-1 LTR transcription by FOXP3.

Prior work has implicated viral protein R (Vpr) in the arrest of human immunodeficiency virus type 1 (HIV-1)-infected cells in the G2 phase of the cell cycle, associated with increased viral replication and host cell apoptosis. We and others have recently shown that virion infectivity factor (Vif ) also plays a role in the G2 arrest of HIV-1-infected cells. Here, we demonstrate that, paradoxically, at early time points postinfection, Vif expression blocks Vpr-mediated G2 arrest, while deletion of Vif from the HIV-1 genome leads to a marked increase in G2 arrest of infected CD4 T-cells. Consistent with this increased G2 arrest, T-cells infected with Vif-deleted HIV-1 express higher levels of Vpr protein than cells infected with wild-type virus. Further, expression of exogenous Vif inhibits the expression of Vpr, associated with a decrease in G2 arrest of both infected and transfected cells. Treatment with the proteasome inhibitor MG132 increases Vpr protein expression and G2 arrest in wild-type, but not Vif-deleted, NL4-3-infected cells, and in cells cotransfected with Vif and Vpr. In addition, Vpr coimmunoprecipitates with Vif in cotransfected cells in the presence of MG132. This suggests that inhibition of Vpr by Vif is mediated at least in part by proteasomal degradation, similar to Vif-induced degradation of APOBEC3G. Together, these data show that Vif mediates the degradation of Vpr and modulates Vpr-induced G2 arrest in HIV-1-infected T-cells.

Objective: Characterization of the effects of HIV-1 infection and apoptosis on cellular and viral gene expression., Methods: Flow cytometry was used to analyze infection and apoptosis concurrently in HIV-1IIIB-infected CEM-SS T cells. Suppression subtractive hybridization (SSH) was applied to cells from different time points of infection to construct subtracted complementary DNA (cDNA) libraries. Differential screening and Northern blots confirmed differential gene expression and these genes were sequenced and compared with database., Results: T cells undergo apoptosis at early stages of HIV-1IIIB infection (days 5-7 post-infection). Surprisingly, cells begin to recover after day 9 and by day 18 almost all infected cells are viable, even though they maintain the same level of infection. By SSH, differential gene expression profiles between day 7 and day 18 after HIV-1IIIB infection were characterized. SSH yielded two subtracted cDNA libraries; differential screening of the subtracted cDNA libraries suggested that 200 out of 864 colonies were highly expressed at their respective time points. DNA sequence analysis identified specific apoptosis-related genes, HIV-1 viral genes, and other candidate genes of interest. Northern blot analysis confirmed that some of these genes were expressed predominantly at the 'apoptotic' or 'non-apoptotic' time points., Conclusions: Known and novel cellular gene products have been identified that are directly (or inversely) correlated with apoptosis and may regulate cell death in HIV-1 infection. These results provide a framework for functional studies on the differentially expressed genes and may suggest novel therapeutic approaches for treatment of HIV-1-infected individuals.

The chemokine receptor, CXCR4, serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus (HIV). Binding of either the CXC-chemokine, stromal-derived factor 1 alpha (SDF-1 alpha), or a CXCR4 antagonist, AMD3100, to CXCR4 inhibits infection of CD4(+) T cells by T-tropic HIV-1, although only SDF-1 alpha triggers T-cell signaling cascades. We have previously demonstrated that ligation of CD4 by T-cell tropic HIV-1 NL4-3 induces metalloproteinase-dependent L-selectin (CD62L) shedding on resting CD4(+) T cells. However, the role of CXCR4 in HIV-induced L-selectin shedding is unclear. Here, we show that L-selectin shedding induced by HIV-1 NL4-3 is completely reversed by AMD3100, but not SDF-1 alpha, although SDF-1 alpha alone does not induce L-selectin shedding. These results indicate that engagement of both CD4 and CXCR4 is required for HIV-induced shedding of L-selectin on primary resting CD4(+) T cells.

Groundbreaking research has led to an understanding of some of the pathogenic mechanisms of HIV-1 infection. Surprisingly, an unanswered question remains the mechanism(s) by which HIV-1 inactivates or kills T cells. Our goals are to define candidate T cell signaling cascades altered by HIV infection and to identify mechanisms whereby HIV-infected cells escape the apoptosis triggered by this aberrant signaling. In earlier work, we found that HIV reprograms healthy T cells to self-destruct by a process called apoptosis. We asked whether apoptosis occurs in organs of infected people and made a surprising discovery-this cell death occurs predominantly in healthy bystander cells and only rarely in infected cells. We hypothesize that HIV may be doubly diabolical-healthy T cells are killed in HIV infection, while infected cells resist killing. Thus, the virus protects its viral factory and allows HIV to turn the cell into a "Trojan Horse," with the virus in hiding or "latent." In this review, we discuss the role of viral and cellular proteins in HIV induced T cell anergy and death. We also discuss mechanisms by which HIV may protect infected T cells from apoptosis. These studies will yield new insights into the pathogenesis of AIDS, identify cellular targets that regulate HIV-1 infection, and suggest novel therapeutic approaches to cure HIV infection.

Gene transfer into primary human CD4 T lymphocytes is a critical tool in studying the mechanism of T cell-dependent immune responses and human immunodeficiency virus-1 (HIV-1) infection. Nucleofection® is an electroporation technique that allows efficient gene transfer into primary human CD4 T cells that are notoriously resistant to traditional electroporation. Despite its popularity in immunological research, careful characterization of its impact on the physiology of CD4 T cells has not been documented. Herein, using freshly-isolated primary human CD4 T cells, we examine the effects of Nucleofection® on CD4 T cell morphology, intracellular calcium levels, cell surface activation markers, and transcriptional activity. We find that immediately after Nucleofection®, CD4 T cells undergo dramatic morphological changes characterized by wrinkled and dilated plasma membranes before recovering 1h later. The intracellular calcium level also increases after Nucleofection®, peaking after 1h before recovering 8h post transfection. Moreover, Nucleofection® leads to increased expression of T cell activation markers, CD154 and CD69, for more than 24h, and enhances the activation effects of phytohemagglutinin (PHA) stimulation. In addition, transcriptional activity is increased in the first 24h after Nucleofection®, even in the absence of exogenous stimuli. Therefore, Nucleofection® significantly alters the activation state of primary human CD4 T cells. The effect of transferred gene products on CD4 T cell function by Nucleofection® should be assessed after sufficient resting time post transfection or analyzed in light of the activation caveats mentioned above. [ABSTRACT FROM AUTHOR]

Abstract: Although HIV-1 (HIV) replicates poorly in non-dividing CD4 lymphocytes, resting T cells contribute to the latent reservoir. The γc-related cytokines reverse this block to HIV infection; however, the molecular mechanisms controlling this process are not understood. We asked whether the γc-cytokine regulated transcription factor, signal transducer and activator of transcription 5 (STAT5), activates HIV transcription. We identified three regions in the long terminal repeat (LTR) as close matches to the STAT5 consensus-binding site and show that STAT5 binds the LTR during HIV infection. Expression of Janus kinase 3 (JAK3) or STAT5 in primary human CD4 T cells activated LTR transcription, while transactivation-incompetent dominant-negative STAT5 inhibited JAK3-induced LTR activity and infection of activated HIV-producing CD4 T-cells. In addition, overexpression of STAT5 increased virus production in unstimulated primary T cells – both the number of p24+ cells and their level of p24 production – suggesting that STAT5 promotes a permissive state for HIV infection. These data may have implications for regulation of latency and therapeutic strategies for control of HIV disease. [Copyright &y& Elsevier]