Murine leukemia virus (MLV) replication monitored with fluorescent proteins.
BACKGROUND
Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity.


RESULTS
We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events.


CONCLUSIONS
Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.
Sliva
Katja
K
Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str, 42-44, 60596 Frankfurt/Main, Germany. schba@pei.de
Erlwein
Otto
O
Bittner
Alexandra
A
Schnierle
Barbara S
BS
D002460:Cell Line; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D009052:Leukemia Virus, Murine / Q000302:isolation & purification / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein