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Foamy viruses are nonpathogenic retroviruses that offer unique opportunities for gene transfer into various cell types including hematopoietic stem cells. We used a simian foamy virus type 1 vector (SFV-1) containing a LacZ reporter gene with a titer of 1-5 x 10(6) viral particles/ml that was free of replication-competent retrovirus to transduce human umbilical cord blood CD34+ cells. Transduced CD34+ cord blood cells were transplanted into NOD/SCID mice and plated in serum-free methylcellulose culture to determine the transduction efficiency of human hematopoietic progenitor cells. A transduction efficiency of about 20% was obtained. At 6-10 weeks posttransplantation, human hematopoietic cell engraftment and marking were determined. Marrow from transplanted mice demonstrated human cell engraftment by the presence of human (CD45+) cells containing both CD19+ lymphoid and CD33+ myeloid cells. Serial sampling of NOD/SCID bone marrow revealed the presence of 6.7-14.0% CD45+ cells at 6 weeks posttransplant as compared to 3.6-27.2% CD45+ cells at 9-10 weeks posttransplant. Human progenitors examined from NOD/SCID bone marrow cells 9 weeks posttransplant revealed from 7.4 to 25.9% of the colonies exhibiting X-gal staining. Our study demonstrates the ability of a simian foamy virus vector to transduce the SCID-repopulating cell and offers a promising new gene delivery system for use in hematopoietic stem cell gene therapy.

Current retroviral vectors based on murine leukemia virus (MuLV) are unable to efficiently transduce nondividing cells. Lentiviruses, such as the human immunodeficiency virus 1 (HIV-1) are efficient at transducing nondividing, growth-arrested, and post-mitotic cells, but due to complex safety considerations, they may have limited potential for human clinical gene transfer. For this reason, alternatives to MuLV and HIV-1 vectors need to be explored. In this paper, we have found that simian foamy virus vector (SFV-1) containing a CMV-LacZ expression cassette is able to efficiently transduce multiple cell types of various species that include epithelial, lymphoid, and hematopoietic-derived human cell lines and fibroblast cell lines of several species. Previously it was reported that foamy virus replication is cell cycle dependent (P. D. Bieniasz, R. A. Weiss, and M. O. McClure, 1995. J. Virol. 69, 7295-7299). However, others studies demonstrated nuclear import of viral DNA in arrested cells (A. Saibi, F. Puvion-Dutilleul, M. Schmid, J. Peries, and H. d. The 1997. J. Virol. 71, 1155-1161). Here, we show efficient LacZ transduction by SFV-1 vectors in several chemically arrested cell lines and terminally differentiated human neurons. SFV-1 vector can transduce cell lines arrested in G1/S phase of the cell cycle by aphidicolin treatment with similar efficiencies to that of dividing cells. The terminally differentiated human neural cell line, NT2N, was transduced with 30-50% efficiency, corroborating our data obtained with the arrested cell lines. To further examine whether the SFV-1 vector can efficiently deliver a gene into clinically important cells for gene therapy, we transduced primary human peripheral blood cells (PBLs) in the presence and absence of phytohemagglutanin (PHA) stimulation. We observed 81% transduction efficiency in non-stimulated PBLs and 87% in PHA-stimulated PBLs with vector infection carried out twice in 8 hours intervals at a multiplicity of infection of 1. Together, these data indicate that SFV-1 based retroviral vectors may provide a safe, efficient alternative to current onco- and lentiviral vectors for gene transfer in cells from a broad spectrum of lineages across species boundaries., (Copyright 2001 Academic Press.)

Biological isolates of feline immunodeficiency virus (FIV) cause a relative expansion of activated single-positive CD8(+) (SP CD8(+)) lymphocytes within the thymus of infected cats. In this study, thymic SP CD8(+) lymphocytes were analyzed from cats inoculated as neonates with a pathogenic molecular clone of FIV, JSY3, which was previously derived from the wild-type biological isolate FIV(NCSU-1) (NCSU-1). Four cats were inoculated intraperitoneally with NCSU-1 and compared with 11 cats inoculated with JSY3. Five control cats matched in litter and age were administered an intraperitoneal sham inoculum. Between 12 and 16 weeks postinoculation, interferon-gamma (IFN-gamma) mRNA was quantified by RT-PCR in freshly isolated thymocytes and peripheral blood mononuclear cells (PBMCs). The quantity of IFN-gamma mRNA was increased more than 10-fold in thymocytes and PBMCs of 13 of 13 FIV-inoculated cats as compared with the sham-inoculated controls. IFN-gamma mRNA coenriched with magnetically sorted CD8(+) PBMCs and single-positive (SP) CD8(+) thymocytes. Cells expressing IFN-gamma mRNA were located within the thymic perivascular zone, along the corticomedullary junction, and adjacent to lymphoid follicles. The expansion of thymic SP CD8(+) cells was associated with an increase in CD8alpha(+)/beta(neg) and CD8alpha(+)/beta(lo) phenotypes, the latter population resembling a previously reported memory/effector peripheral blood cell with FIV suppressor activity. From these data we conclude that JSY3 and NCSU-1 induce similar phenotypic changes in thymic and peripheral blood CD8(+) cells. Thus, JSY3 is pathogenic for the thymus in vivo and will be useful for defining determinants of the CD8(+) cell response in this pediatric AIDS model.

The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Substitutions on an imidazolylmethyl-AMBA-methionine template gave a highly potent and cell-active inhibitor.

A series of highly potent, structurally novel, non-nucleoside RT inhibitors has been described. Low nanomolar concentrations of 5-chloro-3-(phenylsulfonyl)-indole-2-carboxamide (1) inhibit the HIV-1 RT enzyme in vitro and HTLVIIIb viral spread in MT-4 human T-lymphoid cells. Good oral bioavailability was observed in rhesus monkeys upon oral dosing of 1 as a suspension in methocel. When compared to other non-nucleoside inhibitors (e.g. 15-18), 1 possesses improved inhibitory potency with respect to the wild-type RT, as well as the K103N and Y181C mutant enzymes. Additional studies within this class of inhibitors are in progress.

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.

The actions of atrial natriuretic factor (ANF) on the vascular wall are diverse and show a profound regional heterogeneity. ANF is a potent relaxant of aortic smooth muscle, a response which is associated with activation of particulate guanylate cyclase and elevation in tissue levels of cyclic GMP. However, many large and small muscular arteries and most veins are unresponsive to the peptide. The regional vascular heterogeneity may be due to an altered distribution of high affinity receptors and (or) alterations in the coupling of receptor activation to elevations in cyclic 3',5'-guanosine monophosphate (cGMP). Species differences exist in the structural requirements for receptor activation as well as the effects of infused ANF on peripheral resistance. Although the relaxation to ANF in vitro does not require an intact endothelium, endothelial cells contain multiple receptor subtypes for ANF. Differences amongst tissues and (or) species in the receptor profile for ANF may, in part, explain some of heterogeneity in responsiveness to ANF.

The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.