Yves Engelborghs

LEDGF/p75 Is Essential for Nuclear and Chromosomal Targeting of HIV-1 Integrase in Human Cells

We have reported that human immunodeficiency virus type 1 (HIV-1) integrase (IN) forms a specific nuclear complex with human lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75) protein. We now studied the IN-LEDGF/p75 interaction and nuclear import of IN in living cells using fusions of IN and LEDGF/p75 with enhanced green fluorescent protein and far-red fluorescent protein HcRed1. We show that both the N-terminal zinc binding domain and the central core domains of IN are involved in the interaction with LEDGF/p75. Both domains are essential for nuclear localization of IN as well as for the association of IN with condensed chromosomes during mitosis. However, upon overexpression of LEDGF/p75, the core domain fragment of IN was recruited to the nuclei and mitotic chromosomes with a distribution pattern characteristic of the full-length protein, indicating that it harbors the main determinant for interaction with LEDGF/p75. Although the C-terminal domain of IN was dispensable for nuclear/chromosomal localization, a fusion of the C-terminal IN fragment with enhanced green fluorescent protein was found exclusively in the nucleus, with a diffuse nuclear/nucleolar distribution, suggesting that the C-terminal domain may also play a role in the nuclear import of IN. In contrast to LEDGF/p75, its alternative splice variant, p52, did not interact with HIV-1 IN in vitro and in living cells. Finally, RNA interference-mediated knock-down of endogenous LEDGF/p75 expression abolished nuclear/chromosomal localization of IN. We conclude, therefore, that the interaction with LEDGF/p75 accounts for the karyophilic properties and chromosomal targeting of HIV-1 IN.

Novel Inhibitors of HIV-1 Integration

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). The current strategy for the treatment of HIV infection is called Highly Active Antiretroviral Therapy (HAART) and is based on cocktails of drugs that are currently approved by the Food and Drug Administration. These drugs include compounds that target the viral entry step and the enzymes reverse transcriptase or protease. The introduction of HAART has dramatically changed the landscape of HIV disease. Death from AIDS-related diseases has been reduced significantly since HAART came into use. Nevertheless it is not clear how long clinical benefit will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting other steps of the viral replication cycle may increase the potency of inhibition and delay resistance development. HIV integrase is an essential enzyme in the HIV life cycle and is an attractive target for new drug development. Despite years of intensive research, only two classes of compounds that inhibit integration have been identified until now, namely the diketo acids and the pyranodipyrimidines. In this review we will point to new potential antiviral targets related to retroviral integration that are amenable to drug development. We will describe the pitfalls of currently used integrase assays and propose new strategies and technologies for the discovery of HIV integration inhibitors. Furthermore, we will describe the two classes of integrase inhibitors and discuss their antiviral activity, molecular mechanism of anti-HIV action and the selection of HIV resistance against these drugs.

Identification of Authentic Inhibitors of HIV-1 Integration

Current strategies for the treatment of human immunodeficiency virus (HIV) infection are based on cocktails of drugs that target the viral entry step and the enzymes reverse transcriptase or protease. At present, the clinical benefit of this combination therapy for HIV-infected patients is considerable, although it is not clear how long this effect will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting additional steps of the viral replication cycle may increase the potency of inhibition and prevent significant resistance development. During HIV replication, integration of the viral genome into the cellular chromosome is an essential step catalyzed by the viral integrase. Although HIV integrase is an attractive target for antiviral therapy and the focus of intensive research, to date only two classes of compounds that selectively inhibit HIV integration have been identified, namely the diketo acids and the pyranodipyrimidines. In this review we address the question why it has proven so difficult to find potent and selective integrase inhibitors; we point to potential pitfalls in defining an inhibitor as an authentic integrase inhibitor and we propose new strategies and new technologies for the discovery of genuine HIV integration inhibitors. For the diketo acids and the pyranodipyrimidines we will discuss in detail the antiviral activity, the molecular mechanism of anti-HIV action, the in vitro HIV resistance development and the clinical perspectives.