Quantitation of retroviral-mediated transfer using luciferase in living and lysed cells.

We have developed a murine retroviral vector containing an improved luciferase gene for the study of retroviral gene transfer and expression in living or lysed cells. We used a cytosolic form of luciferase gene (luc+) with transcriptional enhancements that yielded greater expression levels. The luc+ gene was subcloned into the retroviral plasmids pDON-AI, in which almost the entire U3 region has been replaced with the heterologous human cytomegalovirus immediate-early promoter A stable ecotropic and amphotropic retrovirus-producing cell line was generated with a titer 1 x 10(6) cfu/mL.

Role of the cytoplasmic tail of ecotropic moloney murine leukemia virus Env protein in fusion pore formation.

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion.

Membrane destabilization assay based on potassium release from liposomes.

Inorganic ions are highly suitable markers for monitoring release of the inner content of liposomes. In the present study, a potassium (K(+)) selective electrode was used to evaluate the rate of K(+) release from large unilamellar vesicles (LUV). The developed method is highly sensitive, reproducible and inexpensive.

Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein.

The cytoplasmic tail of the immature Moloney murine leukemia virus (MoMuLV) envelope protein is approximately 32 amino acids long. During viral maturation, the viral protease cleaves this tail to release a 16-amino-acid R peptide, thereby rendering the envelope protein fusion competent. A series of truncations, deletions, and amino acid substitutions were constructed in this cytoplasmic tail to examine its role in fusion and viral transduction.