Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells.

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

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.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

HIV-1 reverse transcription. A termination step at the center of the genome.

During HIV-1 reverse transcription, the plus-strand of viral DNA is synthesized as two discrete segments. We show here that synthesis of the upstream segment terminates at the center of the genome after an 88 or 98 nucleotide strand displacement of the downstream segment, initiated at the central polypurine tract. Thus, the final structure of unintegrated linear HIV-1 DNA includes a central plus-strand overlap. In vitro reconstitution using only purified reverse transcriptase with appropriate DNA hybrids gave rise to efficient and accurate termination, which was dramatically amplified in the context of strand displacement. Mutation of the sequence immediately upstream of the termination sites almost completely abolished termination both in infected cells and in vitro. This mutation profoundly impaired replication of HIV-1. We conclude that proper central plus-strand termination, mediated by a novel cis-active termination sequence, is a key step in HIV-1 replication.

Kinetic analysis of HIV-1 early replicative steps in a coculture system.

During an acute human immunodeficiency virus (HIV) infection in vitro, different forms of unintegrated viral DNA accumulate in target cells. They include linear full-length HIV DNA molecules, which are the precursors of the integrated provirus, and two types of circular molecules (with one or two LTRs), whose role and mode of formation are not fully understood. To evaluate the intracellular fate of HIV unintegrated DNA, and to follow the formation of the two types of circular DNA molecules, the nuclear transport of viral DNA, and its integration in host cell DNA, we have designed a "DNA chase" assay. This assay is based on cocultivation of persistently HIV-1-infected H9 cells with uninfected MT4, allowing rapid accumulation of viral DNA, which is then blocked by addition of AZT. In this highly efficient, synchronous, one-step cycle infection system, HIV linear DNA can be detected on Southern blots as early as 4 hr after the start of the coculture. Subsequently, viral DNA that had been synthesized before the addition of AZT could be "chased," establishing that almost all linear DNA molecules are rapidly transported to the nucleus, where they are either processed into the two types of circles or integrated. We could estimate that from the number of viral DNA molecules synthesized in 6 hr in this system, at least a third will become integrated and another third will circularize within 24 hr.

HIV-derived vectors for therapy and vaccination against HIV.

Despite being at the origin of one of the world's most devastating public health concerns, the Human Immunodeficiency Virus (HIV) has properties that can be harnessed for therapeutic purposes. Indeed, the ability of HIV to efficiently deliver its genome into the nuclear compartment makes it an ideal vector for gene delivery into target cells. The design of so-called HIV-derived vectors, or more generally lentiviral vectors (LVs), consists in keeping only the parts of the virus that ensure efficient nuclear delivery while entirely removing all coding sequences that contribute towards the replication and pathogenesis of the virus: as a result, the vector genome is composed of less than 10% of the original virus genome and exclusively cis-active sequences. Proteins required for the formation of the lentivector particles and for the early steps of viral replication (including Gag- and Pol-derived proteins) are provided in trans. HIV-derived vectors are thus non-replicative virus shells that deliver genes of interest into target cells with high efficiency. Undoubtedly, there is a hopeful twist of fate in our fight against AIDS, which consists in using these vectors to achieve gene therapy and vaccination against HIV itself. This review summarises the current generation of LVs with a special focus on vaccine applications against AIDS. Preclinical data are very encouraging and efforts are ongoing to optimise these vectors, to increase their safety and improve their immunogenicity.

A haplotype of the human CXCR1 gene protective against rapid disease progression in HIV-1+ patients.

Chemokines and their receptors are key factors in the onset and progression of AIDS. Among them, accumulating evidence strongly indicates the involvement of IL-8 and its receptors, CXCR1 and CXCR2, in AIDS-related conditions. Through extensive investigation of genetic variations of the human CXCR1-CXCR2 locus, we identified a haplotype of the CXCR1 gene (CXCR1-Ha) carrying two nonsynonymous single nucleotide polymorphisms, CXCR1_300 (Met to Arg) in the N terminus extracellular domain and CXCR1_142 (Arg to Cys) in the C terminus intracellular domain. Transfection experiments with CXCR1 cDNAs corresponding to the CXCR1-Ha and the alternative CXCR1-HA haplotype showed reduced expression of CD4 and CXCR4 in CXCR1-Ha cells in human osteosarcoma cells as well as in Jurkat and CEM human T lymphocytes. Furthermore, the efficiency of X4-tropic HIV-1(NL4-3) infection was significantly lower in CXCR1-Ha cells than in CXCR1-HA cells. The results were further confirmed by a series of experiments using six HIV-1 clinical isolates from AIDS patients. A genetic association study was performed by using an HIV-1(+) patient cohort consisting of two subpopulations of AIDS with extreme phenotypes of rapid and slow progression of the disease. The frequency of the CXCR1-Ha allele is markedly less frequent in patients with rapid disease onset than those with slow progression (P = 0.0003). These results provide strong evidence of a protective role of the CXCR1-Ha allele on disease progression in AIDS, probably acting through modulation of CD4 and CXCR4 expression.

Fusion from without directed by human immunodeficiency virus particles.

Fusion from without is the process through which particles of some enveloped viruses can direct fusion of target cells in the absence of viral replication. We demonstrate here that human immunodeficiency virus (HIV) particles can efficiently promote fusion from without. Using HeLa-CD4 cells carrying a Tat-inducible lacZ gene, we observed syncytia as early as 6 h after exposure to HIV particles, before HIV gene expression could be detected. Efficient syncytium formation could be obtained when cells were treated with zidovudine, which prevented HIV replication and expression but not cell-cell fusion. Fusion was also observed when cells were exposed to particles of a replication-defective HIV integrase mutant. Fusion from without by HIV particles could be blocked by a monoclonal antibody specific for the V3 loop of the HIV-1 envelope glycoprotein and by soluble CD4. This mechanism of cytopathicity, which can involve cells that do not actively replicate HIV and can be directed by replication-defective particles, could participate in the pathogenicity of the CD4 cell depletion that characterizes HIV infection.

A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract.

The structure of unintegrated human immunodeficiency virus type 1 (HIV-1) DNA from acutely infected human lymphoid cells was analyzed by nuclease S1 cleavage. We observed a unique, discrete single-stranded gap in unintegrated linear DNA molecules, located near the center of the genome. Oligonucleotide primer extension experiments determined that the downstream limit of this gap coincides with the last nucleotide of a central copy of the polypurine tract found in all sequenced lentivirus genomes. Other retroviruses have only one copy of the polypurine tract at the 5' boundary of the 3' long terminal repeat, which has been shown to determine initiation of retroviral DNA plus-strand synthesis. We conclude from our observations that the central repeat of the polypurine tract can create an additional site for plus-strand synthesis initiation in lentiviruses. The central single-stranded gap was not found in circular DNA molecules, the vast majority of them carrying only one long terminal repeat. This finding suggests that the generation of such circular molecules is associated with early DNA ligation events.

A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication.

We recently reported that human immunodeficiency virus type 1 (HIV-1) unintegrated linear DNA displays a discontinuity in its plus strand, precisely defined by a second copy of the polypurine tract (PPT) located near the middle of the genome (P. Charneau and F. Clavel, J. Virol. 65:2415-2421, 1991). This central PPT appears to determine a second initiation site for retrovirus DNA plus-strand synthesis. We show here that mutations replacing purines by pyrimidines in the HIV-1 central PPT, which do not modify the overlapping amino acid sequence, are able to significantly slow down viral growth as they reduce plus-strand origin at the center of the genome. One of these mutations, introducing four pyrimidines, results in a 2-week delay in viral growth in CEM cells and abolishes plus-strand origin at the central PPT. The introduction in this mutant of a wild-type copy of the PPT at a different site creates a new plus-strand origin at that site. This new origin also determines the end of the upstream plus-strand segment, probably as a consequence of limited strand displacement-synthesis. Our findings further demonstrate the role of PPTs as initiation sites for the synthesis of the retroviral DNA plus strand and demonstrate the importance of a second such origin for efficient HIV replication in vitro.

Complementation of murine cells for human immunodeficiency virus envelope/CD4-mediated fusion in human/murine heterokaryons.

Murine cell lines expressing human CD4 are resistant to the fusogenic effect of the human immunodeficiency virus (HIV) envelope. Consequently, they cannot be infected by HIV or form syncytia with HIV envelope-expressing cells. Murine cells could either lack human-specific cofactors necessary for the CD4/envelope-mediated membrane fusion or express inhibitors of this process. To address this question, we have tested the ability of heterokaryons made from CD4-expressing murine cells and human cells to undergo HIV envelope-mediated fusion. We have devised a rapid and specific assay based on the induction of lacZ expression, in which membrane fusion events with HIV-infected cells can be detected by a simple histochemical technique. CD4-positive murine/human heterokaryons, but not murine/simian heterokaryons, were found able to fuse with HIV envelope-expressing cells. In these experiments, the fusion resistant phenotype of murine-CD4 cells could be complemented by human cellular factors.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate.

The beta-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4(+) target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4(+) CCR-5(+) HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the "nonsyncytium-inducing," primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the "syncytium-inducing" HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the beta-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1beta (MIP-1beta) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these beta-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and beta-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

A highly defective HIV-1 group O provirus: evidence for the role of local sequence determinants in G-->A hypermutation during negative-strand viral DNA synthesis.

The sequence of 2350 nucleotides in the env and IN regions of a group O HIV-1 genome which is hypermutated throughout its entirety was compared to the equivalent sequence of a nonhypermutated genome from the same isolate. Almost 30% of G residues were affected by G-->A transitions. As previously reported, transitions occurred mainly at GpA and GpG dinucleotides, with a marked preference for changes of the 5'-proximal G residues in poly(G) stretches. Inspection of the sequences around the hypermutation sites revealed no bias when the mutation was at the 5' G residue of a GpG dinucleotide. In contrast, a preferred context for hypermutation at the 3' G (or at single G residues) could be defined. In addition to a preference for A residues immediately downstream of hypermutated 3' G residues, C residues were underrepresented in these positions. The observed context fits well with a model whereby G-->A mutation occurs by a combination of dislocation mutagenesis at GpA dinucleotides and direct misincorporation of dTTP at the 5' G of GpG dinucleotides. Furthermore, both runs of six G residues present in the polypurine tracts (PPTs) had escaped hypermutation, despite the fact that 95% of runs of three G residues contained at least one G-->A transition. This finding suggests that genomes with hypermutated PPT motifs had been selected against and provides direct evidence that hypermutation occurs during negative-strand DNA synthesis.

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

Effects of the Tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized Tat protein and Tat peptides.

To study the structure relationship of different Tat domains, the full-length Tat protein Tat1-86, the gene product of the first exon Tat1-72 which retains full activity of the protein, and a panel of shorter peptides mimicking different regions of the primary structure of the Tat protein were chemically synthesized by the solid-phase method, using an efficient protocol. Synthetic Tat1-86 and Tat1-72 transactivated beta-galactosidase activity in HeLa cells containing the lacZ gene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tat1-86 and Tat1-72 with the sulfhydryl of cysteine residues free or protected by the acetamidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat1-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat1-48 and Tat38-60 were used simultaneously, a significant transactivation was obtained. This result suggests that both peptide domains are implicated in transactivation, probably by acting at two different sites. This permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation.

Human immunodeficiency virus type 1 Vif- mutant particles from restrictive cells: role of Vif in correct particle assembly and infectivity.

Disruption of the vif gene of human immunodeficiency virus (HIV) type 1 affects virus infectivity to various degrees, depending on the T-cell line used. We have concentrated our studies on true phenotypic Vif- mutant particles produced from CEMx174 or H9 cells. In a single round of infection, Vif- virus is approximately 25 (from CEMx174 cells) to 100 (from H9 cells) times less infectious than wild-type virus produced from these cells or than the Vif- mutant produced from HeLa cells. Vif- virions recovered from restrictive cells, but not from permissive cells, are abnormal both in terms of morphology and viral protein content. Notably, they contain much reduced quantities of envelope proteins and altered quantities of Gag and Pol proteins. Although wild-type and Vif- virions from restrictive cells contain similar quantities of viral RNA, no viral DNA synthesis was detectable after acute infection of target cells with phenotypically Vif- virions. To examine the possible role of Vif in viral entry, attempts were made to rescue the Vif- defect in H9 cells by pseudotyping Vif+ and Vif- HIV particles with amphotropic murine leukemia virus envelope. Vif- particles produced in the presence of HIV envelope could not be propagated when pseudotyped. In contrast, when only the murine leukemia virus envelope was present, significant propagation of Vif- HIV particles could be detected. These results demonstrate that Vif is required for proper assembly of the viral particle and for efficient HIV Env-mediated infection of target cells.

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 human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells.

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34(+) hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).

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 (DC(CD40LT)), or monocyte-conditioned medium (DC(MCM)) being added or not from day 5. Culture day 8 cells exposed to X4 HIV-1(LAI) or R5 HIV-1(Ba-L) were analyzed by semiquantitative R-U5 PCR, which detects total HIV DNA. CXC chemokine receptor 4(low) (CXCR4(low)) CCR5(+) iDC harbored similar viral DNA amounts when exposed to either strain. HIV-1(LAI) entered more efficiently into DC(CD40LT) or DC(MCM) with up-regulated CXCR4. CCR5(low) DC(CD40LT) still allowed entry of HIV-1(Ba-L), whereas CCR5(-) DC(MCM) 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-1(Ba-L) reverse transcription was more efficient than that of HIV-1(LAI), 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-kappa B proteins and SP1 and SP3 transcription factors.