The insertion of an anti-MHC I ScFv into the N-terminus of an ecotropic MLV glycoprotein does not alter its fusiogenic potential on murine cells
Résumé
It is known that targeted infection requires the modification of the viral envelope, in order to render it capable of recognizing and specifically binding to a marker protein of the target cell. We have previously described such a recombinant envelope, which is able to extend the tropism of an ecotropic murine leukemia viruses (MLV) envelope to MHC I-expressing human cells. Although, this envelope was very efficient in binding human cells, it yielded very low infection titers. Our attempts to improve these yields by the additional cloning of a variety of spacers in the proximity of the single-chain variable fragment (ScFv) moiety did not significantly influenced human titers, although some alterations on murine titers were observed. To examine whether these low yields represent a decreased fusion capacity of the recombinant envelopes, we performed an assay which allowed the direct comparison between the fusiogenicity of the wild-type (w/t) and the chimeric envelopes. No fusiogenicity of the chimeric envelopes was observed when chimera-expressing cells were co-cultured with human cells. The inability of the chimeras to induce fusion after binding of the ScFv moiety to its ligand may explain, in part, the low infection titers on human cells. However, the several-fold differences observed between the titers of the w/t envelope and the various chimeras on murine cells were not reflected on their fusiogenic potentials, which were all in the same order of magnitude. Our results demonstrate that the binding of the ScFv moiety to its ligand induces no fusion, albeit its insertion into the envelope does not alter the intrinsic fusiogenic ability of the latter. Induction of fusion results from the binding of the envelope to the ecotropic receptor, without being directly proportional to its binding affinity. Chimeras with different infection titers on murine cells yielded similar syncytia counts after their binding to the ecotropic receptor.
Mots clés
Insertional Rats Tumor Cells
Murine/genetics/*metabolism/physiology Membrane Fusion/*physiology Mice Mutagenesis
Animals COS Cells Cercopithecus aethiops Glycoproteins/genetics/*metabolism Histocompatibility Antigens Class I/*immunology Humans Immunoglobulin Fragments/genetics/*metabolism Immunoglobulin Variable Region/genetics/*metabolism Leukemia Virus
Cultured Viral Envelope Proteins/genetics/*metabolism