Pseudotype-Independent Nonspecific Uptake of Gammaretroviral and Lentiviral Particles in Human Cells

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. Voelkel and colleagues use fluorescently labeled Gag proteins of murine leukemia virus (MLV) and HIV-1 to tag viral particles and test whether ecotropic, vesicular stomatitis virus (VSV)-G, or MLV pseudotypes, or “bald” particles lacking Env, can be internalized by human cells. Viral particles were detected in the endosomal/lysosomal compartment irrespective of their pseudotype and the potential availability of suitable receptors. All subsequent steps of the life cycle, including those promoting the endosomal/lysosomal release of functional retroviral proteins and mRNA, required the presence of Env and its receptor. Collectively, their findings support the notion that fusion, and not binding or uptake, represents the critical step in the selective targeting of retroviral vector particles in human cells.