Characterization of recombination events leading to the production of an ecotropic replication-competent retrovirus in a GP plus envAM12-derived producer cell line

Replication-competent retrovirus (RCR) was identified in a GP+envAM12-deriv ed producer cell, containing the MFG-S-Neo retroviral vector, using a marke r rescue assay. Studies were undertaken to determine the origin and structu re of this RCR. Receptor interference assays demonstrated that the virus wa s pseudotyped with an ecotropic envelope. Molecular analysis demonstrated t he presence of a MoMLV ecotropic env recombinant where the neomycin resista nce gene of the MFG-S-Neo vector was replaced by MoMLV ecotropic env. Addit ional recombinants linking the retroviral pol gene to neo and the neo gene to MoMLV env were also identified. A full-length MoMLV retroviral genome wa s detected by nested PCR in the contaminated amphotropic producer cells and in cells infected with its supernatant Unexpectedly, this was also present in the GP+E86 packaging cells together with a previously undescribed envel ope construct possessing a full 5' and 3' LTR, although these cells were co nsistently negative for the presence of RCR. These anomalies in the GP+E86 packaging cell line result in increased homology with the MFG-S-Neo vector, leading to an increased risk for the production of RCR. Our findings point to a need for increased vigilance when using these packaging lines to gene rate replication-defective retrovirus. (C) 2000 Academic Press.