Replication-competent retrovirus produced by a 'split-function' third generation amphotropic packaging cell line

We report the discovery, on routine screening, of a replication-competent retrovirus (RCR) produced from a third generation amphotropic packaging cell line, GP+envAM12. In this line, the gag-pol and env helper genes are located on separate plasmids to minimise the chances of recombination events that may lead to RCR formation. Plasmid pBabeNeo was transfected into GP+envAM12 to obtain a producer line. Supernatant from this line was used to infect two lines, B16 and 1735-puro, and stable colonies which were obtained were pooled. Supernatants from both these lines were able to transfer the neo gene to fresh cells through several passages, indicating the presence of helper virus. Rescue of puro from 1735-puro cells, which contain the pBabePuro provirus, was also achieved. Mobilisation of lacZ from TELCeB6 cells, which contain the MFGnlslacZ provirus, was also possible. The RCR was able to transfer lacZ to ampli-GPE cells, an ecotropic packaging cell line, but transfer to fresh GP+envAM12 cells was minimal, demonstrating receptor interference of the RCR by the amphotropic envelope. These observations strongly suggest that a RCR with an amphotropic envelope arose in the GP+envAM12-pBabeNeo producer line. To our knowledge, this is the first report of RCR arising from routine use of GP+envAM12 cells or any similar third generation packaging line and has important implications for the use of such lines in human gene therapy protocols.