Integrase-LEDGF/p75 complex triggers the formation of biomolecular condensates that modulate HIV-1 integration efficiency in vitro.

The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO).

The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton.

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end-directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex.

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence.

Biological and Structural Analyses of New Potent Allosteric Inhibitors of HIV-1 Integrase.

HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles. Herein, we describe a new series of INLAIs based on a benzene scaffold that display antiviral activity in the single digit nanomolar range.

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures.

The HIV-1 Integrase C-Terminal Domain Induces TAR RNA Structural Changes Promoting Tat Binding.

Recent evidence indicates that the HIV-1 Integrase (IN) binds the viral genomic RNA (gRNA), playing a critical role in the morphogenesis of the viral particle and in the stability of the gRNA once in the host cell. By combining biophysical, molecular biology, and biochemical approaches, we found that the 18-residues flexible C-terminal tail of IN acts as a sensor of the peculiar apical structure of the trans-activation response element RNA (TAR), interacting with its hexaloop. We show that the binding of the whole IN C-terminal domain modifies TAR structure, exposing critical nucleotides.

A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor.

The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles.

Unstable Protein Purification Through the Formation of Stable Complexes.

Purification of proteins containing disordered regions and participating in transient complexes is often challenging because of the small amounts available after purification, their heterogeneity, instability, and/or poor solubility. To circumvent these difficulties, we set up a methodology that enables the production of stable complexes in large amounts for structural and functional studies. In this chapter, we describe the methodology used to establish the best cell culture conditions and buffer compositions to optimize soluble protein production and their stabilization through protein complex formation.

Translational regulation of APOBEC3G mRNA by Vif requires its 5'UTR and contributes to restoring HIV-1 infectivity.

The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5'-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently.

The HDAC6/APOBEC3G complex regulates HIV-1 infectiveness by inducing Vif autophagic degradation.

Background: Human immunodeficiency virus type 1 (HIV-1) has evolved a complex strategy to overcome the immune barriers it encounters throughout an organism thanks to its viral infectivity factor (Vif), a key protein for HIV-1 infectivity and in vivo pathogenesis. Vif interacts with and promotes "apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3G" (A3G) ubiquitination and subsequent degradation by the proteasome, thus eluding A3G restriction activity against HIV-1.

Results: We found that cellular histone deacetylase 6 (HDAC6) directly interacts with A3G through its C-terminal BUZ domain (residues 841-1,215) to undergo a cellular co-distribution along microtubules and cytoplasm.

HIV-1 replication and the cellular eukaryotic translation apparatus.

Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms.

APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag).

The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription.

Purification of nuclear poly(A)-binding protein Nab2 reveals association with the yeast transcriptome and a messenger ribonucleoprotein core structure.

Nascent mRNAs produced by transcription in the nucleus are subsequently processed and packaged into mRNA ribonucleoprotein particles (messenger ribonucleoproteins (mRNPs)) before export to the cytoplasm. Here, we have used the poly(A)-binding protein Nab2 to isolate mRNPs from yeast under conditions that preserve mRNA integrity. Upon Nab2-tandem affinity purification, several mRNA export factors were co-enriched (Yra1, Mex67, THO-TREX) that were present in mRNPs of different size and mRNA length.

Characteristics and prognosis of patients with angiographic stent thrombosis: comparison between drug-eluting and bare-metal stents.

Introduction: Conflicting data exist on the risk of stent thrombosis with drug-eluting stents (DES) versus bare-metal stents (BMS). Little is known about the potential different characteristics and outcomes of DES versus BMS thrombosis.

Objective: To compare the characteristics, timing and outcomes of patients with angiographic stent thrombosis according to type of stent implanted.

Cardiac events after low osmolar ionic or isosmolar nonionic contrast media utilization in the current era of coronary angioplasty.

Objectives: Our study aimed to compare the isosmolar nonionic dimer iodixanol and the low osmolar ionic agent ioxaglate in the current era of percutaneous coronary intervention (PCI), using clopidogrel, enoxaparine, direct stenting, and drug eluting stent.

Background: Previous studies have suggested an association between thrombus-related events and type of contrast media.

Methods: Our prospective single-center study included 498 consecutive patients who were assigned to receive either iodixanol (n = 231) or ioxaglate (n = 267).

Epstein-Barr virus mRNA export factor EB2 is essential for intranuclear capsid assembly and production of gp350.

Most human herpesviruses, including Epstein-Barr virus (EBV), express a protein which functions primarily as an mRNA export factor. Previously, we deleted the gene for the Epstein-Barr virus mRNA export factor EB2 from the EBV genome and then introduced the mutated genome into 293 cells. Using a transcomplementation assay in which ectopic expression of the transcription factor EB1/ZEBRA was sufficient to induce the EBV productive cycle, we showed that Ori-Lyt-dependent replication of the EBV DNA occurs in the absence of EB2, indicating that EB2 is not essential for the expression and export of early mRNAs.

Long-term outcome after coronary angioplasty in renal transplant and hemodialysis patients.

In order to determine how renal transplantation modifies in hospital and long-term outcome after coronary angioplasty, we compared dialysis and renal transplant patients with control patients without renal failure. Seventy-five consecutive dialysis patients (group D) and 37 renal transplant patients (group T) undergoing coronary angioplasty, were compared with two control groups (groups control D and control T, respectively) matched 1:1 with groups D and T for clinical and angiographic characteristics. The mean follow-up was 50 months.

[Sleep-related breathing disorders and normal or high-tension glaucoma: 35 patients with polysomnographic records].

Purpose: To look for early stage glaucoma symptoms in patients referred for sleep-related breathing disorders or sleep apnea syndrome.

Patients And Method: Thirty-five patients between October 2001 and April 2002 consecutively admitted for polysomnographic evaluation were investigated. Each patient had complete ophthalmologic examination before the nocturnal record.

[Absence of nephro-protective effect of acetylcysteine in patients with chronic renal failure investigated by coronary angiography].

Recent studies have suggested that an oral dose of acetylcysteine could play a prophylactic role in the prevention of nephrotoxicity from iodine contrast media in patients affected by chronic renal failure. Between June 2001 and September 2002 we selected 120 patients with a basal plasma creatinine level greater than 1.36 mg/dl investigated by coronary angiography.

Comparison of short- and long-term outcomes of coronary angioplasty in patients with and without diabetes mellitus and with and without hemodialysis.

In-hospital and long-term outcomes after coronary angioplasty in 28 dialysis diabetic and 84 dialysis nondiabetic patients were compared with clinical outcomes after coronary angioplasty in 28 nondialysis diabetic and 84 nondialysis, nondiabetic patients matched according to clinical and angiographic characteristics. The rate of angiographic success in diabetic dialysis patients was high and similar in the 4 groups. The risk of 4-year cardiac death and nonfatal myocardial infarction was higher in dialysis diabetics than in dialysis nondiabetics (odds ratio [OR] 1.

Safety, efficacy, and cost advantages of combined coronary angiography and angioplasty.

Aim: The safety and efficacy of ad hoc PTCA has been previously reported and this approach is performed in many angioplasty centers as a routine procedure. The aim of this study is to examine whether this approach reduces the length, and cost of hospital stay.

Methods And Results: To determine the hospital costs we studied 2,440 PTCAs over 11 years in our institution (1990-2000).