Simple criterion for selection of flavonoid compounds with anti-HIV activity.

Flavonoid compounds represent an important natural source of antiretrovirals for AIDS therapy due to their significant anti-HIV-1 activity and low toxicity. Here we propose a simple theoretical criterion to discriminate active from inactive flavonoids that is suitable for rapid in silico screening of flavonoid libraries, and selection and optimization of lead compounds with anti-HIV-1 activity.

Identification of the LEDGF/p75 binding site in HIV-1 integrase.

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part.

Concise review: therapeutic strategies for Parkinson disease based on the modulation of adult neurogenesis.

Parkinson disease (PD) is a progressive neurodegenerative disorder affecting millions of people worldwide. To date, treatment strategies are mainly symptomatic and aimed at increasing dopamine levels in the degenerating nigrostriatal system. Hope rests upon the development of effective neurorestorative or neuroregenerative therapies based on gene and stem cell therapy or a combination of both.

Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication.

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.

The central DNA flap of the human immunodeficiency virus type 1 is important for viral replication.

Reverse transcription of the human immunodeficiency virus type 1 is characterized by the formation of a DNA flap at the center of the viral cDNA in between the central polypurine tract (cPPT) and the central termination sequence (CTS). The importance of the DNA flap for HIV-1 replication has been questioned, whereas its importance for lentiviral vector performance is well accepted. To investigate this controversy, we re-evaluated the importance of the DNA flap for HIV-1 replication.

Parkin protects against neurotoxicity in the 6-hydroxydopamine rat model for Parkinson's disease.

Loss-of-function mutations in the PARK2 gene are the major cause of early onset familial Parkinson's disease. The gene product, parkin, is an E3 ligase of the ubiquitin-proteasome pathway involved in protein degradation. Dopaminergic neuron loss may result from the toxic accumulation of parkin substrates, suggesting a key role for parkin in dopaminergic neuron survival.

Comparison of lentiviral vector titration methods.

Background: Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression.

Noninvasive monitoring of long-term lentiviral vector-mediated gene expression in rodent brain with bioluminescence imaging.

Gene transfer into the central nervous system is an emerging therapeutic strategy for a range of neurological diseases, including neurodegeneration. This approach would benefit from imaging technologies that could determine the extent, magnitude, and duration of transgene expression. We have used bioluminescence imaging (BLI) to image lentiviral vector-mediated gene transfer into the mouse brain.

Lentiviral vectors mediate efficient and stable gene transfer in adult neural stem cells in vivo.

Modulation of adult neurogenesis may offer new therapeutic strategies for various brain disorders. In the adult mammalian brain the subventricular zone (SVZ) of the lateral ventricle is a region of continuous neurogenesis. Lentiviral vectors stably integrate into dividing and nondividing cells, in contrast to retroviral vectors, which integrate only into dividing cells.

Transient and stable knockdown of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle of human immunodeficiency virus.

After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication.

Non-invasive imaging of neuropathology in a rat model of alpha-synuclein overexpression.

Parkinson's disease is a neurodegenerative disorder affecting the dopaminergic neurons in the substantia nigra. Aggregation of alpha-synuclein appears to play a central role in the pathogenesis. Novel animal models for neurodegeneration have been generated by lentiviral vector-mediated locoregional overexpression of disease-associated genes in the adult brain.

The aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E.

Cellular co-factors of HIV-1 integration.

To achieve productive infection, the reverse transcribed cDNA of human immunodeficiency virus type 1 (HIV-1) is inserted in the host cell genome. The main protein responsible for this reaction is the viral integrase. However, studies indicate that the virus is assisted by cellular proteins, or co-factors, to achieve integration into the infected cell.

Pharmacophore-based design of HIV-1 integrase strand-transfer inhibitors.

Using a training set of diketo-like acid HIV-1 integrase (IN) strand-transfer inhibitors, a 3D pharmacophore model was derived having quantitative predictive ability in terms of activity. The best statistical hypothesis consisted of four features (one hydrophobic aromatic region, two hydrogen-bond acceptors, and one hydrogen-bond donor) with r of 0.96.

Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology.

A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al.

Upscaling of lentiviral vector production by tangential flow filtration.

Background: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods.

Methods: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation.

Hematopoietic stem cell-based gene therapy against HIV infection: promises and caveats.

Stem cell-based gene therapy of HIV infection aims at inhibiting HIV replication and the progression to AIDS by the introduction of antiviral genes in primitive hematopoietic stem cells (HSC). Ideally, after differentiation into mature blood cells, these antiviral genes should create a host-cell population that is resistant to HIV infection. Although the current gene therapy clinical trials established the safety and provided proof-of-principle for gene therapy of HIV-1 infection, the overall results have been disappointing, and many issues still remain to be resolved before this approach can be efficiently used against HIV infection.

HIV-1 integration: an interplay between HIV-1 integrase, cellular and viral proteins.

To achieve a productive infection, the reverse transcribed cDNA of the human immunodeficiency virus type 1 (HIV-1) has to be inserted in the host cell genome. The main protein required to accomplish this reaction is the virally encoded integrase. In vitro, the recombinant integrase is capable of catalyzing the two subsequent reactions of the integration process, namely the 3' processing followed by the strand transfer, without other viral and/or cellular proteins.

Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication.

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication.

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.

The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding.

We have previously shown that the p75 isoform of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) interacts tightly with human immunodeficiency virus (HIV)-1 integrase (IN) and is essential for nuclear targeting of this protein in human cells (Cherepanov, P., Maertens, G., Proost, P.

Positional effects of the central DNA flap in HIV-1-derived lentiviral vectors.

During reverse transcription of the human immunodeficiency virus type 1 (HIV-1), a 90- or 99-nucleotide long DNA flap is formed at the centre of the viral cDNA. The presence of a central DNA flap in lentiviral vectors improves transduction efficiency significantly. We analysed the stimulation of lentiviral vector transduction by a DNA flap present at ectopic positions in the viral cDNA.

Evaluation of the activity of HIV-1 integrase over-expressed in eukaryotic cells.

Since the integration of viral DNA in the host genome is an essential step in the replication cycle of HIV-1, an active search for inhibitors of the integration step is ongoing. Our laboratory has been working on the development of a cellular integration system. Such a system would be helpful in the study of the HIV-1 integration process and, eventually, could be used in the search for new inhibitors that selectively interfere with HIV integration.

Multiple mutations in human immunodeficiency virus-1 integrase confer resistance to the clinical trial drug S-1360.

Objectives: Study of HIV-1 resistance development to the diketo analogue S-1360, the first HIV-1 integrase strand transfer inhibitor that has entered clinical development.

Design: HIV-1(IIIB) was passaged in cell culture in the presence of increasing concentrations of S-1360 (IIIB/S-1360(res)).

Methods: The IIIB/S-1360(res) strains selected for 30, 50 and 70 passages in the presence of S-1360 were evaluated genotypically by sequencing analysis and phenotypically using the MT-4/MTT assay.

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.