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The effective entry of retroviruses into target cells depends on the presence of viral envelope (Env) proteins and cognate cellular receptors, such as the murine cationic amino acid transporter-1 (mCAT-1) for the ecotropic murine leukemia virus (MLV-E). Here, we examined whether human cells internalize MLV-E or other retroviral pseudotypes irrespective of the presence of a specific receptor. Using fluorescently tagged Gag to monitor viral internalization, and treating cells with chloroquine or bafilomycin A1, we show that endocytosis is the main pathway for productive transduction with ecotropic particles, but endocytosis of retroviral particles itself does not depend on a suitable receptor or Env. Nonspecific endosomal uptake and lysosomal degradation occurred with all "illegitimate" envelope-receptor combinations tested: MLV particles pseudotyped with the ecotropic envelope or measles virus H and F proteins as well as "ecotropic" or "bald" HIV-1 particles. Kinetic studies in cell lines and primary human T lymphocytes showed the persistence of Gag-GFP signals for more than 10 days after exposure to retroviral vector particles, even in the absence of a suitable receptor. Further studies testing the Gag-mediated transfer of protein or retroviral mRNA revealed that nonspecific endocytosis prevented the release of functional particle-associated proteins and nucleic acids into the cytosol. We conclude that receptor-targeted retroviral particles are unlikely to escape nonspecific cellular uptake unless appropriate protective principles are discovered. Conversely, as lysosomal degradation was found to inactivate mRNA and proteins embedded into retroviral particles, receptor targeting is a useful strategy for both transient and permanent cell modification by retrovirus-like particles.

The regulated expression of therapeutic genes may become crucial in gene therapy when their constitutive expression interferes with cell fate in vivo. The efficient regulation of transgene expression requires tightly controlled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its application requires the introduction of two components into the target cell genome: the tet-responsive transactivator and the regulated expression cassette. In order to facilitate the usage of the tet-system for approaches in gene therapy, both components have to be transferred by a single vector, thus eliminating the preselection of transactivator positive cells. Published "all-in-one" vectors for regulated transgene expression display a relatively low signal-to-noise ratio, resulting in regulatory windows of around 500-fold even in selected clones. In this study, we show that a modified vector architecture combined with the introduction of new tet-responsive promoters, Ptet, improved the dynamic range of such all-in-one vectors to levels up to 14,000-fold for viral and 25,000-fold for nonviral transfer vectors in nonclonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improved regulation was the result of a strong reduction of background expression in the off-state, even if cells were transduced at high multiplicity of infection, while induction remained at high levels. In addition, the results indicated that successful regulation of gene expression in different target cells depended on vector architecture as well as the choice of the Ptet-promoter.