Omar K. Haffar

Differential expression and sequence-specific interaction of karyopherin α with nuclear localization sequences

The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins α, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1α are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins α to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.

Oxadiazols: a New Class of Rationally Designed Anti-Human Immunodeficiency Virus Compounds Targeting the Nuclear Localization Signal of the Viral Matrix Protein

ABSTRACT Despite recent progress in anti-human immunodeficiency virus (HIV) therapy, drug toxicity and emergence of drug-resistant isolates during long-term treatment of HIV-infected patients necessitate the search for new targets that can be used to develop novel antiviral agents. One such target is the process of nuclear translocation of the HIV preintegration complex. Previously we described a class of arylene bis(methylketone) compounds that inhibit HIV-1 nuclear import by targeting the nuclear localization signal (NLS) in the matrix protein (MA). Here we report a different class of MA NLS-targeting compounds that was selected using computer-assisted drug design. The leading compound from this group, ITI-367, showed potent anti-HIV activity in cultures of T lymphocytes and macrophages and also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue. The virus carrying inactivating mutations in MA NLS was resistant to ITI-367. Analysis by real-time PCR demonstrated that the compound specifically inhibited nuclear import of viral DNA, measured by two-long terminal repeat circle formation. Evidence of the existence of this mechanism was provided by immunofluorescent microscopy, using fluorescently labeled HIV-1, which demonstrated retention of the viral DNA in the cytoplasm of drug-treated macrophages. Compounds inhibiting HIV-1 nuclear import may be attractive candidates for further development.

Nuclear translocation as a novel target for anti-HIV drugs

During recent years, remarkable progress has been achieved in the treatment of patients infected with HIV. This progress involves not only the improvement of previously known drugs but also the introduction of new classes of anti-HIV agents. Currently, drugs targeting virus entry, reverse transcription, integration and maturation are either in clinical use or in the late stages of clinical development. Nonetheless, the high mutation rate of the virus and toxicity of the drugs, which become problematic during prolonged treatment regimens characteristic of anti-HIV therapy, drive the necessity to produce new drugs that will allow physicians to keep the virus at bay in patients on lifelong anti-HIV therapy. Ideally, such drugs would target a new step in the HIV life cycle, thus avoiding crossresistance with older compounds. One such new target for anti-HIV therapy is nuclear translocation – a process critical for HIV replication. In this article, the authors will review recent literature on the mechanisms of HIV nuclear import and will describe compounds that inhibit this step of HIV replication.

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

Complete activation of peripheral blood T cells requires both T‐cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus‐1 (HIV‐1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28‐mediated signal transduction using reverse transcriptase‐mediated polymerase chain reaction (RT‐PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV‐1 tat gene but not the interleukin‐2 (IL‐2) gene. Viral inductiondid not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H‐7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL.

Small molecule inhibitor of HIV-1 nuclear import suppresses HIV-1 replication in human lymphoid tissue ex vivo : a potential addition to current anti-HIV drug repertoire

Despite recent progress in anti-HIV therapy, which has to do mainly with introduction of protease inhibitors into clinical practice, drug toxicity and emergence of drug-resistant isolates during the long-term treatment of the patients necessitates search for new drugs that can be added to currently used components of a multi-drug cocktail in highly active anti-retroviral therapy (HAART). Recently, we described a class of arylene bis(methylketone) compounds that inhibit nuclear import of HIV-1 pre-integration complexes and suppress viral replication in macrophages and PBMC in vitro. In this report, we demonstrate that one of these compounds, CNI-H1194, inhibited HIV-1 replication in primary lymphoid tissue ex vivo. The compound did not antagonize the activity of currently used anti-HIV drugs that inhibit viral reverse transcriptase or protease. These results suggest that arylene bis(methylketone) compounds might be a valuable addition to HAART.