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.

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.

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 virioncontaining 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.

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 HP1␣. The CTIP2 protein harbors two Tat Regulation of human immunodeficiency virus type 1 (HIV-1) gene transcription is governed by a complex interplay between chromatin-associated proviral DNA, host cell proteins, and the virus-encoded transactivator protein, Tat. In the immediateearly phase of HIV infection, cellular transcription factors activate transcription from the viral long terminal repeat (LTR) (for a review, see references 24 and 32). This leads to the accumulation of the viral protein Tat that leads to a potent increase in transcription and is required for viral replication and a high viral load (for a review, see reference 37). The ability of Tat to function as a transcriptional activator is mediated by multiple interactions with cellular proteins and requires the concerted action of Tat and upstream nuclear factors that bind to the Sp1 and B region of the LTR (for reviews, see references 16 and 17). Tat forms a ternary complex with the coactivators P/CAF and p300 which helps Tat activate transcription of integrated viral DNA and derepress the HIV-1 chromatin structure in response to histone acetylation (3). Mechanisms that inhibit HIV-1 LTR expression are largely unexplored. Recent studies have shown that Tat activation can be inhibited by the overexpression of the host factors YY1 and LSF, which recruit histone deacetylase 1 to the LTR (20).We have previously reported that Tat also interacts and cooperates with an orphan member of the nuclear receptor superfamily, the chicken ovalbumin upstream promoter transcription factor (COUP-TF) that together with Sp1 activates HIV-1 LTR-driven transcription (34,35). Members of the COUP-TF family were recently shown to bind to novel and related zinc finger proteins and COUP TF-interacting protein 1 (CTIP1) and CTIP2. CTIP1 was found to induce transcriptional silencing by relocating COUP-TF to distinct nuclear structures, possibly associated with heterochromatic regions (1). These studies revealed a novel mechanism for transcriptional repression, by recruitment of a transcription factor to distinct nuclear loci, instead of acting t...

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We present the characterization of two overlapping human transferrin genomic clones isolated from a liver DNA library. The two clones represent a total length of 24 kilobase pairs and code for 70% of the protein. The

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