Véronique Zennou

HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap

HIV-1 and other lentiviruses have the unique property among retroviruses to replicate in nondividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV-1 reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap: the central DNA flap. We show here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import. Wild-type viral linear DNA is almost entirely imported into the nucleus where it integrates or circularizes. In contrast, mutant viral DNA, which lacks the DNA flap, accumulates in infected cells as unintegrated linear DNA, at the vicinity of the nuclear membrane. Consistently, HIV-1 vectors devoid of DNA flap exhibit a strong defect of nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.

The HIV-1 DNA flap stimulates HIV vector- mediated cell transduction in the brain

During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. We recently reported that the DNA flap acts as a cis-active determinant of HIV-1 genome nuclear import. Commonly employed HIV-1–derived vectors (HR vectors) lack the central DNA flap. Here we report that the insertion of this DNA flap sequence into HR vectors (TRIP vectors) improves gene transduction in neural cells, ex vivo and in vivo, in rat brain. When neural cells are exposed to increasing concentrations of TRIP vector particles, transgene expression correlates with the dose of vector. This effect contrasts with the plateau observed when using an HR vector. We further demonstrate that the increase of in vivo transduction efficiency obtained with TRIP vectors is due to the stimulation of their genome nuclear import.

The Maturation of Dendritic Cells Results in Postintegration Inhibition of HIV-1 Replication

Maturation of dendritic cells (DC) is known to result in decreased capacity to produce HIV due to postentry block of its replicative cycle. In this study, we compared the early phases of this cycle in immature DC (iDC) and mature DC (mDC) generated from monocytes cultured with GM-CSF and IL-4, trimeric CD40 ligand (DCCD40LT), or monocyte-conditioned medium (DCMCM) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1LAI or R5 HIV-1Ba-L were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4low (CXCR4low) CCR5+ iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1LAI entered more efficiently into DCCD40LT or DCMCM with up-regulated CXCR4. CCR5low DCCD40LT still allowed entry of HIV-1Ba-L, whereas CCR5− DCMCM displayed reduced permissivity to this virus. Comparing amounts of late (long terminal repeat (LTR)-gag PCR) and total (R-U5 PCR) viral DNA products showed that HIV-1Ba-L reverse transcription was more efficient than that of HIV-1LAI, but was not affected by DC maturation. Southern blot detection of linear, circular, and integrated HIV DNA showed that maturation affected neither HIV-1 nuclear import nor integration. When assessing virus transcription by exposing iDC to pNL4-3.GFP or pNL4-3.Luc viruses pseudotyped with the G protein of vesicular stomatitis virus (VSV-G), followed by culture with or without CD40LT or MCM, GFP and luciferase activities decreased by 60–75% in mDC vs iDC. Thus, reduced HIV replication in mDC is primarily due to a postintegration block occurring mainly at the transcriptional level. We could not relate this block to altered expression and nuclear localization of NF-κB proteins and SP1 and SP3 transcription factors.

Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy

A central triple‐stranded DNA structure created during HIV‐1 reverse transcription, the central flap, acts as a cis‐active nuclear import determinant of the HIV‐1 DNA genome. Insertion of the sequences responsible for formation of the central DNA flap into an HIV‐1‐derived vector strongly enhances its transduction efficiency.

Long-Term Expression of β-Glucuronidase by Genetically Modified Human Neural Progenitor Cells Grafted into the Mouse Central Nervous System

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease caused by beta-glucuronidase (beta-glu) deficiency. This deficiency results in the lysosomal accumulation of glycosaminoglycans in all tissues and affects a wide range of organs, including the central nervous system (CNS). Gene transfer is a promising approach to therapy for MPS VII because it allows extensive delivery of the enzyme to the affected tissues. We studied neurotransplantation of primary human cells to supply beta-glucuronidase to the CNS. Human neural progenitor cells (HNPC) were amplified and cotransduced with two lentiviral vectors, one encoding the green fluorescent protein and the other the human beta-glu. We show that these cells strongly expressed both transgenes in culture. When grafted into the mouse striatum, HNPC differentiated into neurons and astrocytes and expressed the two transgenes for at least 6 months. This study therefore paves the way for the treatment of MPS VII by long-term delivery of the appropriate enzyme.

Transplantation of Human Adult Astrocytes: Efficiency and Safety Requirements for an Autologous Gene Therapy

Ex vivo gene therapy is emerging as a promising approach for the treatment of neurodegenerative diseases and central nervous system (CNS) trauma. We have shown previously that human adult astrocytes can be expanded in vitro and can express various therapeutic transgenes (Ridet et al. [ 1999 ] Hum. Gene Ther. 10:271–280; Serguera et al. [ 2001 ] Mol. Ther. 3:875–881). Here, we grafted normal and lentivirally‐modified human adult astrocytes into the striatum and spinal cord of nude mice to test whether they are suitable candidates for ex vivo CNS gene therapy. Transplanted cells survived for at least 2 months (longest time analyzed) and sustained transgene expression. Importantly, the absence of proliferating cell nuclear antigen (PCNA) staining, a hallmark of cell division, ascertains the safety of these cells. Thus, adult human astrocytes are a promising tool for human CNS repair; they may make autologous ex vivo gene transfer feasible, thereby avoiding the problems of immunological rejection and the side effects of immunosuppressors.