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

Abstract

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.

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

“…9, right). Such pseudotype-independent nonspecific uptake of gammaretroviral and lentiviral particles into human cells has been reported before also for particles pseudotyped with the MV vaccine strain GPs (32). Taken together, these data can be used for proposing a model of PTV interaction with APCs after inoculation and subsequent presentation of peptides via MHC-I or MHC-II (Fig.…”

“…This is in line with our strategy to release the cargo protein into the target cell's cytosol by insertion of the additional HIV-1 protease cleavage motif, as described before (22) and as demonstrated in our study by cytosolic transfer and subsequent function of Cre recombinase in the respective indicator cell lines. Moreover, Ova protein transfer also occurred exclusively in receptorpositive cells, as assessed by immunoblot analysis after removal of surface-bound virions by citric acid buffer treatment (32). Both experiments indicate cytoplasmic protein transfer and exclude passive surface adhesion of PTV particles.…”

“…PCR products were ligated into pCR2.1-TOPO (Life Technologies), resulting in pCR2.1HIV1_MA_P_Ova, and the absence of mutations was confirmed by sequencing (Eurofins MWG Operon, Ebersberg, Germany). To enable directed cloning, the 3= end of pCR2.1HIV1_MA_P_Ova was elongated by a 1.64-kb gag fragment obtained from NsiI-cleaved pcDNA3.GPϩGFP.4xCTE (22,32) to yield pCR2.1-gag-Ova.NsiI. The desired sequence from the resulting plasmid was cloned via ClaI and SbfI with the intended insert orientation into pcDNA3.GPϩGFP.4xCTE to yield pcHIV1_MA_P_Ova.…”

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells

“…Lentiviral vector plasmids pRRL.PPT.EFS.EGFP.pre [50–52] and pRRL.PPT.SFFV.DsRedExpress.pre [53] were referred to as LV and LV.DsRed, respectively. Gammaretroviral vectors pSERS11.EFS.EGFP.pre [52] and pSF91.DsRedExpress [53] were termed GV and GV.DsRed.…”

“…Gammaretroviral vectors pSERS11.EFS.EGFP.pre [52] and pSF91.DsRedExpress [53] were termed GV and GV.DsRed. Except for the LTR-driven GV.DsRed, all vectors had SIN designs with deletions of the viral U3 regions (∆U3), instead containing the internal EFS promoter (short version/250 bp fragment of promoter/enhancer sequences from the human elongation factor 1 alpha gene) or, for LV.DsRed, the SFFV promoter (promoter/enhancer sequences from spleen focus forming virus).…”