A Replication-Competent Retrovirus Arising from a Split-Function Packaging Cell Line Was Generated by Recombination Events between the Vector, One of the Packaging Constructs, and Endogenous Retroviral Sequences

Abstract

Previously we reported the presence of a replication-competent retrovirus in supernatant from a vector-producing line derived from a widely used split-function amphotropic packaging cell line. Rigorous routine screening of all retroviral stocks produced in our laboratory has not, previously or since, indicated the presence of such a virus. Replication-competent retroviruses have never previously been used in our laboratory, and stringent screening of all routinely used cell lines has not revealed the presence of any helper viruses. Therefore, it is highly unlikely that this virus represents an adventitious cross-contaminant or had been imported unknowingly with our cell line stocks. PCR studies with DNA from infected cell lines and Northern blot analysis and reverse transcriptase PCR with RNA from infected cells suggest that the helper virus arose by recombination events, at sites of partial homology, between sequences in the vector, one of the packaging constructs, and endogenous retroviral elements. These recombinations were not present in stocks of the packaging cell line or in an initial stock of the vector-producing line, indicating that these events occurred while the vector-producing line was being passaged for harvest of supernatant stocks.

In Vivo Rescue of a Silent tax -Deficient Bovine Leukemia Virus from a Tumor-Derived Ovine B-Cell Line by Recombination with a Retrovirally Transduced Wild-Type tax Gene

“…We suggest that the high sequence homology (97.1%) between the BLV variant 1 (LTaxSN)-and variant 2 (YR2)-derived tax sequences favors crossovers, resulting in the emergence of new recombinant strains. This implies that distinct RNAs (an 8.4-kb BLV-derived RNA for YR2 and a 3.8-kb MoMLV-derived RNA for LTaxSN) can be copackaged, confirming that there is little influence of RNA origin and size on the efficiency of retrovirus recombination (10,32).…”

pa317 Retrovirus Packaging Cells

“…Theoretically this should reduce the rate of helper virus generation compared with that of PA317 cells, in which only two recombination events are required. However, production of replication-competent retrovirus has been documented in split packaging cells [20,21], so even this system does not guarantee safety from helper virus contamination. Compounding this situation is the existence of many endogenous retroviral elements in cells that can provide substrates for recombination to generate replication-competent viruses.…”

“…Compounding this situation is the existence of many endogenous retroviral elements in cells that can provide substrates for recombination to generate replication-competent viruses. Indeed, the helper virus that arose from the split packaging cells described above involved recombination between endogenous retroviral elements, the 3Ј end of the retroviral vector, and only one of the packaging DNAs, that encoding the Env protein [21]. Thus while the split packaging design and other theoretical improvements in packaging cells have occurred since the development of PA317 cells, many factors contribute to the frequency of helper virus generation in vector-producing packaging cells, and all cell lines must be continuously evaluated for potential helper virus production.…”

Progress with retroviral gene vectors

“…Also in this case, three recombination events are required to restore a replication competent virus. However, replication competent virus generated by a third generation packaging cell line has been described [15,16].…”

“…Such as sequences range from infectious and replication competent retroviruses (found in many murine cells), to defective viruses, virus like particles and retroviral elements [29]. As already mentioned, these sequences can recombine with the packaging functions and/or the vector in packaging cell lines and generate a replication competent virus [16]. One additional possibility is that transcripts produced by viral endogenous sequences are packaged by the vectors and transferred to the target cells.…”