The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors.

The human endogenous retrovirus type W (HERV-W) family includes proviruses with intact protein-coding regions that appear to be under selection pressure, suggesting that some HERV-W proviruses may remain active in higher primates. The envelope glycoprotein (Env) encoded by HERV-W is highly fusogenic, is naturally expressed in human placental syncytiatrophoblasts, and has been reported to function as a superantigen in lymphocyte cultures. Recent evidence suggested that HERV-W Env can mediate syncytium formation by interacting with the human sodium-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) (J.

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-activating polypeptide.

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both.

Tumor antigen-specific induction of transcriptionally targeted retroviral vectors from chimeric immune receptor-modified T cells.

High-level systemic delivery of viral vectors to tumors has proved problematic as a result of immune neutralization, nonspecific adhesion, and clearance of circulating viral particles. Some cell types localize to tumors in response to particular biological properties associated with tumor growth. Their use to deliver viral vectors to tumors would allow precious viral stocks to be protected until they can be released at high local concentrations.

High levels of transduction of human dendritic cells with optimized SIV vectors.

As major antigen-presenting cells and effectors in the maintenance of tolerance, dendritic cells (DCs) are key cells of the immune system and can thus be envisioned to have roles in immunotherapy strategies. We, and others, previously showed that simian immunodeficiency virus (SIV)-derived lentiviral vectors were able to deliver a gene into human differentiated DCs. We describe here the upgrading of the SIV vector system and the improvements of the transduction protocol, which allowed us to transduce more than 90% of human monocyte-derived DCs.

MuLV packaging systems as models for estimating/measuring retrovirus recombination frequency.

Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in the formation of recombinant viruses. Using sensitive RT-PCR assays, we have investigated human and murine gene therapy packaging cell lines for the incorporation of endogenous retrovirus transcripts into murine leukaemia virus (MLV) vector particles and whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were packaged in particles produced by the murine AM12 packaging system.

Post-mitotic, differentiated myotubes efficiently produce retroviral vector from hybrid adeno-retrovirus templates.

We have examined the ability of proliferating myoblasts and post-mitotic, differentiated myotubes to produce retroviral vector using hybrid adeno-retroviral vectors as templates. We show that production of retroviral vector from myoblasts peaks 48 h after adenoviral infection at 4.8 x 10(4) cfu/ml and is scarcely detectable by 96 h.

Retargeting gene delivery using surface-engineered retroviral vector particles.

Retroviral vectors with the capacity to deliver transgenes to specific tissues are expected to be of great value for various gene transfer applications in vivo. Initial attempts to modify vector host-range by the insertion of ligands on their surface glycoproteins have frequently failed, essentially owing to the impairment of the fusogenicity of the vector particles bound to the targeted cell-surface molecules. Several strategies aimed to recover the fusogenic activity of surface-engineered vector particles have recently been explored and have given rise to novel concepts in the field.

Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors.

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting).

Concentration of viral vectors by co-precipitation with calcium phosphate.

Background: The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks.

Activation of membrane fusion by murine leukemia viruses is controlled in cis or in trans by interactions between the receptor-binding domain and a conserved disulfide loop of the carboxy terminus of the surface glycoprotein.

Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive.

T cell-specific expression from Mo-MLV retroviral vectors containing a CD4 mini-promoter/enhancer.

Background: Gene therapy of various immunological disorders will greatly benefit from improved retroviral vectors (RVs) with T cell specificity. Such vectors can be designed by placing a gene of therapeutic interest under the control of tissue-specific transcriptional elements. However, low titers and loss of specificity are frequently encountered with tissue-specific vectors.

Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells.

We describe the generation and the characterization of new lentiviral vectors derived from SIVmac251, a simian immunodeficiency virus (SIV). A methodical approach was used to engineer both efficient and safe packaging constructs allowing the production of SIV viral core proteins. SIV-vectors encoding GFP (green fluorescent protein) were generated as VSV-G-pseudotyped particles upon transient expression of the vector construct and helper functions in 293 cells.

Development of minimal lentivirus vectors derived from simian immunodeficiency virus (SIVmac251) and their use for gene transfer into human dendritic cells.

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5' cytomegalovirus early promoter, the 5' viral sequences up to the 5' end of gag required for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3' long terminal repeat (LTR).

Definition of an amino-terminal domain of the human T-cell leukemia virus type 1 envelope surface unit that extends the fusogenic range of an ecotropic murine leukemia virus.

Murine leukemia viruses (MuLV) and human T-cell leukemia viruses (HTLV) are phylogenetically highly divergent retroviruses with distinct envelope fusion properties. The MuLV envelope glycoprotein surface unit (SU) comprises a receptor-binding domain followed by a proline-rich region which modulates envelope conformational changes and fusogenicity. In contrast, the receptor-binding domain and SU organization of HTLV are undefined.

A hyperfusogenic gibbon ape leukemia envelope glycoprotein: targeting of a cytotoxic gene by ligand display.

An important goal in cancer gene therapy is the development of novel targeted cytotoxic genes. The observation that transfection of a GaLV envelope glycoprotein lacking an R peptide into human cells results in considerable cell-cell fusion and subsequent cell death prompted us to explore the potential for using this fusogenic membrane glycoprotein (FMG) as a targeted cytotoxic gene. As proof of principle, we therefore displayed epidermal growth factor (EGF) on the N terminus of GaLV envelope glycoproteins both with and without an R peptide (GaLV R+ and GaLV R-).

Myocardial injury-induced fibroblast proliferation facilitates retroviral-mediated gene transfer to the rat heart in vivo.

Background: Efficient and stable transfer of therapeutic DNA into injured myocardium would be an initial step towards a genetic treatment aimed at myocardial repair after myocardial infarction. Proliferating cardiac fibroblasts in the healing myocardium could be a compelling target for retroviral infection. We evaluated the feasibility of direct in vivo gene transfer into injured myocardium using a high-titer, stable retroviral vector.

Modifying the host range properties of retroviral vectors.

Gene therapy protocols would be greatly facilitated by the availability of targetable injectable vectors which could deliver genes in vivo to specific target cells or to specific disease sites. Efforts to develop such retroviral vectors are therefore a high priority in gene therapy research. In this review, we describe the current state of our understanding of the structure and function of the retroviral envelope glycoprotein complex.

An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor.

A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F.

Incorporation of simian virus 5 fusion protein into murine leukemia virus particles and its effect on the co-incorporation of retroviral envelope glycoproteins.

We describe the generation of murine leukemia virus (MLV) virus particles carrying the paramyxovirus fusion protein F from simian virus 5 (SV5-F). This glycoprotein was expressed in cells providing Moloney MLV (MoMLV) Gag and Pol proteins and a lacZ retroviral vector. SV5-F was correctly expressed, processed, and efficiently incorporated into retroviral particles.

Activation of a cell entry pathway common to type C mammalian retroviruses by soluble envelope fragments.

Mutations that negatively or positively affect the fusion properties of murine leukemia viruses (MLVs) have been found within all subdomains of their SU (surface) and TM (transmembrane) envelope units. Yet, the interrelations between these different regions of the envelope complex during the cell entry process are still elusive. Deletion of the histidine residue of the conserved PHQV motif at the amino terminus of the amphotropic or the ecotropic MLV SU resulted in the AdelH or the MOdelH fusion-defective mutant envelope, respectively.

Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors.

We recently showed that retroviral vectors can be targeted through protease substrate interactions. Infectivity is blocked by a polypeptide fused to the viral envelope glycoprotein (SU) and is restored when a protease cleaves the connecting linker, releasing the inhibitory polypeptide from the viral surface. Protease specificity is achieved by engineering the sequence of the linker.

Retrovirus targeting by tropism restriction to melanoma cells.

Targeted vectors will be necessary for many gene therapy applications. To target retroviruses to melanomas, we fused a single-chain variable fragment antibody (scFv) directed against the surface glycoprotein high-molecular-weight melanoma-associated antigen (HMW-MAA) to the amphotropic murine leukemia virus envelope. A proline-rich hinge and matrix metalloprotease (MMP) cleavage site linked the two proteins.

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope.