Murine leukemia virus (MLV) replication monitored with fluorescent proteins

Katja Sliva,Alexandra Bittner,Barbara S Schnierle,Otto Erlwein

doi:10.1186/1743-422x-1-14

Katja Sliva, Alexandra Bittner + Show 2 more

Open Access

https://doi.org/10.1186/1743-422x-1-14

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Journal: Virology journal	Publication Date: Jan 1, 2004
Citations: 44	License type: cc-by

Affiliation: Georg Speyer Haus, Paul Ehrlich Institut

Abstract

BackgroundCancer 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.ResultsWe 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.ConclusionsFluorescently 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.

Highlights

Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect
We inserted the coding sequences for green fluorescent protein (GFP) into the prolinerich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label murine leukemia virus (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
A replication competent ecotropic MLV variant was generated (GFP-EMO) that had the 53 aas of the epidermal growth factor (EGF) fused to the N-terminus of Env and the GFP sequences inserted into the proline-rich region (PRR) [7]

Summary

Introduction

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, 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. Efficient and long-lasting gene delivery is the major challenge in the development of vectors for gene therapy. Replication-competent retroviruses (RCRs) encoding suicide genes linked via an internal ribosome entry site (IRES) offer a significant advantage over replication-deficient vectors in cancer gene therapy, since they are able to spread efficiently in vivo [1,2,3,4]. The selective delivery of a therapeutic gene by targeting retroviral entry would immensely reduce unfavorable side effects and ease the clinical application of gene therapy. An obvious challenge has (page number not for citation purposes)

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