Evelyne Schaeffer

Human Immunodeficiency Virus Type 1 Nef Functions at the Level of Virus Entry by Enhancing Cytoplasmic Delivery of Virions

ABSTRACT The Nef protein of the type 1 human immunodeficiency virus (HIV-1) plays a key although poorly understood role in accelerating the progression of clinical disease in vivo. Nef exerts several biological effects in vitro, including enhancement of virion infectivity, downregulation of CD4 and major histocompatibility complex class I receptor expression, and modulation of various intracellular signaling pathways. The positive effect of Nef on virion infectivity requires its expression in the producer cell, although its effect is manifested in the subsequent target cell of infection. Prior studies suggest that Nef does not alter viral entry into target cells; nevertheless, it enhances proviral DNA synthesis, arguing for an action of Nef at the level of viral uncoating or reverse transcription. However, these early studies discounting an effect of Nef on virion entry may be confounded by the recent finding that HIV enters cells by both fusion and endocytosis. Using epifluorescence microscopy to monitor green fluorescent protein-Vpr-labeled HIV virion entry into HeLa cells, we find that endocytosis forms a very active pathway for virus uptake. Virions entering via the endocytic pathway do not support productive infection of the host cell, presumably reflecting their inability to escape from the endosomes. Conversely, our studies now demonstrate that HIV Nef significantly enhances CD4- and chemokine receptor-dependent entry of HIV virions into the cytoplasmic compartment of target cells. Mutations in Nef either impairing its ability to downregulate CD4 or disrupting its polyproline helix compromise virion entry into the cytoplasm. We conclude that Nef acts at least in part as a regulator of cytosolic viral entry and that this action contributes to its positive effects on viral infectivity.

Regulation of HIV‐1 gene transcription: from lymphocytes to microglial cells

Transcription is a crucial step for human immunodeficiency virus type 1 (HIV‐1) expression in all infected host cells, from T lymphocytes, thymocytes, monocytes, macrophages, and dendritic cells in the immune system up to microglial cells in the central nervous system. To maximize its replication, HIV‐1 adapts transcription of its integrated proviral genome by ideally exploiting the specific cellular environment and by forcing cellular stimulatory events and impairing transcriptional inhibition. Multiple cell type‐specific interplays between cellular and viral factors perform the challenge for the virus to leave latency and actively replicate in a great diversity of cells, despite the variability of its long terminal repeat region in different HIV strains. Knowledge about the molecular mechanisms underlying transcriptional regulatory events helps in the search for therapeutic agents that target the step of transcription in anti‐HIV strategies.

Compensatory Link between Fusion and Endocytosis of Human Immunodeficiency Virus Type 1 in Human CD4 T Lymphocytes

ABSTRACT Virions of the type 1 human immunodeficiency virus (HIV-1) can enter target cells by fusion or endocytosis, with sharply different functional consequences. Fusion promotes productive infection of the target cell, while endocytosis generally leads to virion inactivation in acidified endosomes or degradation in lysosomes. Virion fusion and endocytosis occur equally in T cells, but these pathways have been regarded as independent because endocytosis of HIV virions requires neither CD4 nor CCR5/CXCR4 engagement in HeLa-CD4 cells. Using flow cytometric techniques to assess the binding and entry of green fluorescent protein (GFP)-Vpr-labeled HIV virions into primary peripheral blood mononuclear cells, we have found that HIV fusion and endocytosis are restricted to the CD4-expressing subset of cells and that both pathways commonly require the initial binding of HIV virions to surface CD4 receptors. Blockade of CXCR4-tropic HIV virion fusion with AMD3100, a CXCR4-specific entry inhibitor, increased virion entry via the endocytic pathway. Similarly, inhibition of endosome acidification with bafilomycin A1, concanamycin A, or NH4Cl enhanced entry via the fusion pathway. Although fusion remained dependent on CD4 and chemokine receptor binding, the endosome inhibitors did not alter surface expression of CD4 and CXCR4. These results suggest that fusion in the presence of the endosome inhibitors likely occurs within nonacidified endosomes. However, the ability of these inhibitors to impair vesicle trafficking from early to late endosomes in some cells could also increase the recycling of these virion-containing endosomes to the cell surface, where fusion occurs. In summary, our results reveal an unexpected, CD4-mediated reciprocal relationship between the pathways governing HIV virion fusion and endocytosis.

COUP-TF and Sp1 interact and cooperate in the transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat in human microglial cells.

We have recently reported that chicken ovalbumin upstream promoter transcription factor (COUP-TF) activates human immunodeficiency virus type 1 (HIV-1) gene transcription in glial and neuronal cells. Here, we have examined the role of COUP-TF in microglial cells, the major target cells for HIV-1 infection in brain. We show that COUP-TF activates gene expression from both the lymphotropic LAI and the macrophage-tropic JR-FL HIV-1 strains. Although COUP-TF binds to the −352/−320 nuclear receptor responsive element of the long terminal repeat, it functions as a transcriptional activator by acting on the −68/+29 minimal promoter. This region is a direct target of transcription factors Sp1 and Sp3. We report the discovery and features of a physical and functional interplay between COUP-TF and Sp1. Our cotransfection experiments provide evidence for a functional synergism between Sp1 and COUP-TF leading to enhanced transcriptional activity of the HIV-1 long terminal repeat through the Sp1 element. In contrast, Sp3 functions as a repressor of Sp1- or COUP-TF-induced activation. We further demonstrate that COUP-TF and Sp1 are capable of physically interacting, via the DNA-binding domain of COUP-TF, in vitro and in the cell. These findings reveal how the novel interplay of Sp1 and COUP-TF families of transcription factors regulate HIV-1 gene expression.

COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415–5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1α, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region.

Recruitment of Tat to Heterochromatin Protein HP1 via Interaction with CTIP2 Inhibits Human Immunodeficiency Virus Type 1 Replication in Microglial Cells

ABSTRACT The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1α. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1α associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1α complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis.

Functional interactions between C/EBP, Sp1, and COUP-TF regulate human immunodeficiency virus type 1 gene transcription in human brain cells.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system (CNS) and plays a direct role in the pathogenesis of AIDS dementia. However, mechanisms underlying HIV-1 gene expression in the CNS are poorly understood. The importance of CCAAT/enhancer binding proteins (C/EBP) for HIV-1 expression in cells of the immune system has been recently reported. In this study, we have examined the role and the molecular mechanisms by which proteins of the C/EBP family regulate HIV-1 gene transcription in human brain cells. We found that NF-IL6 acts as a potent activator of the long terminal repeat (LTR)-driven transcription in microglial and oligodendroglioma cells. In contrast, C/EBPγ inhibits NF-IL6-induced activation. Consistent with previous data, our transient expression results show cell-type-specific NF-IL6-mediated transactivation. In glial cells, full activation needs the presence of the C/EBP binding sites; however, NF-IL6 is still able to function via the minimal −40/+80 region. In microglial cells, C/EBP sites are not essential, since NF-IL6 acts through the −68/+80 LTR region, containing two binding sites for the transcription factor Sp1. Moreover, we show that functional interactions between NF-IL6 and Sp1 lead to synergistic transcriptional activation of the LTR in oligodendroglioma and to mutual repression in microglial cells. We further demonstrate that NF-IL6 physically interacts with the nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF), via its DNA binding domain, in vitro and in cells, which results in mutual transcriptional repression. These findings reveal how the interplay of NF-IL6 and C/EBPγ, together with Sp1 and COUP-TF, regulates HIV-1 gene transcription in brain cells.

CREB and COUP‐tF mediate transcriptional activation of the human immunodeficiency virus type 1 genome in jurkat t cells in response to cyclic AMP and dopamine

Infection of lymphocytes by the human immunodeficiency virus type 1 (HIV‐1) is associated with an increase in intracellular cAMP levels. Recent studies have shown that lymphocytes are able to synthesize and bind the dopamine, known to affect multiple cellular pathways, such as the cAMP pathway. Here we have investigated the molecular mechanisms by which cAMP and dopamine regulate HIV‐1 gene transcription in Jurkat T cells. Transient expression experiments revealed that dopamine and forskolin lead to a synergistic stimulation of long terminal repeat (LTR)‐driven transcription. This action is mediated through the cAMP response element binding (CREB) protein and chicken ovalbumin upstream promoter transcription factor (COUP‐TF). CREB and COUP‐TF act indirectly through the minimal ‐40/+80 and ‐68/+80 LTR region, respectively. We have previously demonstrated that COUP‐TF stimulates HIV‐1 transcription via the ‐68/+29 LTR region without direct DNA binding. Here, gel supershift experiments show that CREB does not directly bind to the ‐45/+85 proximal LTR sequences. Moreover, our data reveal novel functional interactions between COUP‐TF and CREB, which lead to synergistic cAMP‐ and dopamine‐induced transactivation of the HIV‐1 LTR. These findings reveal that dopamine‐induced signals and the cAMP pathway stimulate HIV‐1 gene transcription in lymphocytes by converging to the minimal –68/+80 LTR region, through the transcription factors CREB and COUP‐TF. J. Cell. Biochem. 75:404–413, 1999.

The Nuclear Receptor Chicken Ovalbumin Upstream Promoter Transcription Factor Interacts with HIV-1 Tat and Stimulates Viral Replication in Human Microglial Cells

Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system and plays a direct role in the pathogenesis of AIDS dementia. However, the molecular mechanisms underlying HIV-1 expression in the central nervous system are poorly understood. We have recently reported that the nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of the nuclear receptor superfamily, is an activator of HIV-1 gene transcription. Here, our results show that COUP-TF stimulates HIV-1 transcription in primary cultured human microglial cells, the primary target for HIV-1 infection in brain. Run-on assays indicated that COUP-TF acts on the initiation step of transcription. Results from reverse transcription-polymerase chain reaction and immunocytochemistry analysis further revealed the importance of this factor by demonstrating that overexpression of COUP-TF leads to initiation of viral replication in primary HIV-infected human microglia. In addition, COUP-TF is able to physically interact and cooperate with the viral transactivator Tat. The combination of COUP-TF and Tat leads to NF-κB- and Sp1-independent enhanced transcriptional stimulation.In vitro binding studies showed that COUP-TF interacts with Tat through amino acids within the N-terminal DNA-binding domain of COUP-TF. Amino acids 48–72 in the basic and C-terminal regions of Tat are required for the binding of Tat to COUP-TF. These results suggest that COUP-TF is an essential transcription factor involved in HIV-1 expression in microglia and reveal a novel interplay of Tat and COUP-TF during regulation of viral expression.