| Human immunodeficiency virus-1 (HIV-1) based vectors are a promising approach for gene therapy targeting fetal tissues, however little is known about the role of accessory proteins in HIV-1 based gene transfer vectors in vivo. The impact of HIV-1 accessory proteins on gene transfer efficiency was analyzed in a murine in utero gene transfer model. Semi-quantitative PCR analysis revealed comparable levels of enhanced green fluorescent (EGFP) gene transfer by vectors, prepared in the presence or absence of HIV-1 accessory proteins, Nef, Vif, Vpu, and Vpr, in multiple organs systems, and immunoselected peripheral blood mononuclear cells (PBMCs). The EGFP transgene was detected in both mature myeloid and lymphoid cell lineages, as well as lineage marker negative marrow cells, with both vector preparations at all time points. Analysis of ex vivo expanded bone marrow-derived hematopoietic progenitor cells revealed successful gene transfer in both erythroid and myeloid lineages in the both vector groups, analyzed as early as one month of age.; Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL). In addition to typical retroviral structural and enzymatic gene products, HTLV-1 also encodes unique regulatory and accessory proteins, including a singly-spliced pX open reading frame (ORF) II product p13II. We have demonstrated that proviral clones of HTLV-1, which are mutated in pX ORFII fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13II localizes to mitochondria and reduce cell growth and tumorgenicity in mice, but its function in human lymphocytes remains undetermined. Herein, we analyzed functional properties of Jurkat T cells expressing p13II, using both transient and stable expression vectors. Our data indicated that p13II expressing Jurkat T cells were sensitive to caspase-dependent, ceramide- and Fast-induced apoptosis. p13II expressing Jurkat T cells also exhibited reduced proliferation when cultured at high density. Furthermore, pre-incubation of the p13II expressing cell with a farnesyl transferase inhibitor, which blocks post translational modification of Ras, markedly reduced Fast induced apoptosis, indicating participation of Ras pathway in p13II 's influence of lymphocyte survival. Our data is the first to demonstrate that p13II alters Ras-mediated apoptosis in T-lymphocytes, and reveals a potential mechanism for HTLV-1 to alter lymphocyte proliferation. (Abstract shortened by UMI.)... |
