Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression.

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CD4+ cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5Δ). We report that the chronically HIV-infected U1 cell line expresses STAT5Δ but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5Δ and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF–stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5Δ binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5Δ by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5Δ present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.

The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/ AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4 ؉ T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34 ؉ human stem cells (HSCs). Moreover, we identified the 126-to 170-amino-acid region in the Cterminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. IntroductionAnti-HIV-1 gene therapy is based on the concept that autologous hemopoietic stem cells (HSCs) carrying antiviral transgenes once reinfused into HIV-1 ϩ subjects will naturally expand and differentiate into CD4 ϩ T cells and macrophages, the 2 major HIV-1 cellular hosts. HIV-1-resistant CD4 ϩ T cells and macrophages will then contribute to reconstitute the heavily compromised immune system of AIDS patients. 1 It is manifest that an anti-HIV-1/AIDS gene therapy strategy should affect early step(s) of HIV-1 life cycle, such as viral entry and proviral DNA integration, to be most effective. This will reduce the generation of infected cells from noninfected cells. 1,2 Viral infectivity factor (Vif) is an HIV-1 key protein because it counteracts the action of the cellular anti-HIV-1 restriction factor human APOBEC3G (hA3G) that, in the absence of Vif, has a deadly effect on HIV-1 replication. 3 Vif is therefore an excellent target for the development of new anti-HIV-1/AIDS gene therapy approaches. In this context, we have previously reported that the natural F12-Vif mutant, containing 14 unique amino-acid substitutions, 4 once delivered in CD4 ϩ T lymphocytes by second-generation lentiviral vector (LV), efficiently prevents HIV-1 production. 5 Recently, several Vif functional domains involved in either protein-protein or protein-viral RNA interactions have been identified. [6][7][8][9][10] Of particular interest is the identification of a novel HCCH zinc-coordination motif conserved among primate lentivirus Vifs. [11][12][13] This motif is critical for the interaction of Vif and Cullin5, which is one of the E3 ubiquitin ligase multiprotein complex deputed to hA3G degradation. 14 Gene therapy preclinical studies, aimed at providing safety, feasibility, and efficacy analyses, mainly privilege the use of CD4 ϩ T lymphocytes because these cells are easy to obtain, cultivate, and transduce by viral vectors, and are highly susceptible to HIV-1 infection in vitro. In contrast, primary macrophages are nonproliferating terminally differentiated cells, which are transduced by LVs at efficiency not higher than 30%. [15][16][17][18] Hence, despi...