Jo Vercammen

Overexpression of the Lens Epithelium-Derived Growth Factor/p75 Integrase Binding Domain Inhibits Human Immunodeficiency Virus Replication

ABSTRACT We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.

Development of Resistance against Diketo Derivatives of Human Immunodeficiency Virus Type 1 by Progressive Accumulation of Integrase Mutations

ABSTRACT The diketo acid L-708,906 has been reported to be a selective inhibitor of the strand transfer step of the human immunodeficiency virus type 1 (HIV-1) integration process (D. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, and M. D. Miller, Science 287:646-650, 2000). We have now studied the development of antiviral resistance to L-708,906 by growing HIV-1 strains in the presence of increasing concentrations of the compound. The mutations T66I, L74M, and S230R emerged successively in the integrase gene. The virus with three mutations (T66I L74M S230R) was 10-fold less susceptible to L-708,906, while displaying the sensitivity of the wild-type virus to inhibitors of the RT or PRO or viral entry process. Chimeric HIV-1 strains containing the mutant integrase genes displayed the same resistance profile as the in vitro-selected strains, corroborating the impact of the reported mutations on the resistance phenotype. Phenotypic cross-resistance to S-1360, a diketo analogue in clinical trials, was observed for all strains. Interestingly, the diketo acid-resistant strain remained fully sensitive to V-165, a novel integrase inhibitor (C. Pannecouque, W. Pluymers, B. Van Maele, V. Tetz, P. Cherepanov, E. De Clercq, M. Witvrouw, and Z. Debyser, Curr. Biol. 12:1169-1177, 2002). Antiviral resistance was also studied at the level of recombinant integrase. Single mutations did not appear to impair specific enzymatic activity. However, 3′ processing and strand transfer activities of the recombinant integrases with two (T66I L74M) and three (T66I L74M S230R) mutations were notably lower than those of the wild-type integrase. Although the virus with three mutations was resistant to inhibition by diketo acids, the sensitivity of the corresponding enzyme to L-708,906 or S-1360 was reduced only two- to threefold. As to the replication kinetics of the selected strains, the replication fitness for all strains was lower than that of the wild-type HIV-1 strain.

The Saccharomyces cerevisiae alcohol acetyl transferase Atf1p is localized in lipid particles

The yeast alcohol acetyl transferase I, Atf1p, is responsible for the major part of volatile acetate ester production in fermenting Saccharomyces cerevisiae cells. Some of these esters, such as ethyl acetate and isoamyl acetate, are important for the fruity flavours of wine, beer and other fermented beverages. In order to reveal the subcellular localization of Atf1p and further unravel the possible physiological role of this protein, ATF1::GFP fusion constructs were overexpressed in brewers yeast. The transformant strain showed a significant increase in acetate ester formation, similar to that of an ATF1 overexpression strain, indicating that the Atf1p–GFP fusion protein was active. UV fluorescence microscopy revealed that the fusion protein was localized in small, sphere‐like organelles. These organelles could be selectively stained by the fluorescent dye Nile red, indicating that they contained high amounts of neutral lipids and/or sterols, a specific characteristic of yeast lipid particles. Purification of lipid particles from wild type and ATF1 deletion cells confirmed that the Atf1p–GFP fusion protein was located in these organelles. Furthermore, a clear alcohol acetyl transferase activity could be measured in the purified lipid particles of both wild type and transformed cells. The localization of Atf1p in lipid particles may indicate that Atf1p has a specific role in the lipid and/or sterol metabolism that takes place in these particles. Copyright

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.

Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75

Recently we described the interaction of human immunodeficiency virus type 1 (HIV‐1)1 integrase (IN) with a cellular protein, lens epithelium‐derived growth factor/transcription co‐activator p75 (LEDGF/p75). We now present the study of the diffusion behavior of the three independent domains of IN and LEDGF/p75 using fluorescence correlation microscopy (FCM). We show that diffusion in the cell of the different enhanced green fluorescent protein (EGFP) fusion proteins is described by two components with different fractions and that the average parameters in the nucleus are comparable with those in the cytoplasm. In addition, we demonstrate that specific interaction between EGFP‐fused HIV‐1 IN and LEDGF/p75 results in a shift in diffusion coefficient (D). The opposite shift was observed in an IN‐deletion mutant that does not exhibit LEDGF/p75 binding or in a LEDGF/p75 knock‐down experiment using siRNA. We thus demonstrate that protein–protein interactions can be studied in living cells, using single‐color FCM (scFCM).

Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

Abstract A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore coated beads that mimic the behaviour of multiliganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

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.

Measuring Diffusion in a Living Cell Using Fluorescence Correlation Spectroscopy. A Closer Look at Anomalous Diffusion Using HIV-1 Integrase and its Interactions as a Probe

Fluorescence correlation spectroscopy is a powerful technique for observing the diffusion offluorescent molecules. In buffer solution the autocorrelation analysis of the intensity fluctuations allowsthe calculation of the diffusion coefficient, and therefore the size of the molecule, as well as its interactions.In living cells, however, the situation is more complicated because the viscosity of the medium varies a lotfrom spot to spot. Moreover, the analysis of the autocorrelation curve is not always straightforward andthe chosen analysis method can sometimes be questioned. Autocorrelation curves obtained from living cellscan be fitted equally well with a multicomponent model or with an anomalous diffusion model (Wachsmuthet al., J Mol Biol, 298:677, 2000). The latter model introduces a global anomaly parameterthat describes the heterogeneity of the hindrance of the medium to the diffusing molecules in the cell.By applying the two models on a system of interacting proteins in living cells, we could compare theadvantages and disadvantages of both models. This article will give an introduction to fluorescence correlationspectroscopy as well as HIV-1 integrase, whose protein fragments have been used as a model in thisstudy. HIV-1 integrase has been shown to interact with a cellular protein, called lens epithelium-derivedgrowth factor or LEDGF/p75. This interaction will be used as a starting point for the comparison ofthe two models.