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. 1998 Apr;72(4):2663-70.
doi: 10.1128/JVI.72.4.2663-2670.1998.

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

H Chong  1 , W Starkey, R G Vile
Affiliations

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

H Chong et al. J Virol. 1998 Apr.

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.

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Figures

FIG. 1
FIG. 1
Northern blots of RNA extracted from parental and helper virus-infected cells. RNA was obtained from K1735-puro/neo cells, parental K1735 cells, helper virus-infected NIH 3T3 cells, and parental NIH 3T3 cells. Northern blots of the RNA were probed with 4070A env (top panel). The blot was stripped and probed with neo (middle panel) and then, as a control, probed with the gene for murine glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (bottom panel).
FIG. 2
FIG. 2
Genomic structure of helper virus and the pBabeNeo vector. (a) Schematic diagram of the helper virus genome showing the locations of PCR primers used in this study (not drawn precisely to scale). The subgenomic transcript bearing the env gene is also depicted. s.d., splice donor site; s.a., splice acceptor site. (b) Schematic diagram of the pBabeNeo vector (15). A subgenomic transcript containing neo, driven by the simian virus 40 (SV40) early promoter, would also be obtained.
FIG. 3
FIG. 3
(a) Amplification of an env-3′ LTR fragment by PCR with DNA from helper virus-infected NIH 3T3 cells. (A) As controls for the presence of genomic DNA, PCR was performed with primers for the promoter of the murine tyrosinase gene, with DNA of GP+envAM12 cells and parental and helper virus-infected NIH 3T3 cells. (B) An env-LTR fragment was amplified with primers RV152 plus RV116. A positive signal was obtained only in the reaction with DNA of infected NIH 3T3 cells. (b) Amplification of a 5′ LTR–gag-pol fragment by PCR with DNA from helper virus-infected cells. Primer RV150 corresponds to a sequence in MoMLV LTR, and RV151 is at the pol/env overlap region. PCR with DNA of GP+envAM12 showed two signals, consistent with amplification from the two packaging constructs. The reaction with DNA of infected NIH 3T3 cells showed one signal of approximately 5.7 kb, which was absent in the reaction with parental NIH 3T3 cells. (c) Amplification of a pol-4070A env fragment by PCR with DNA from helper virus-infected cells. For PCR with primers RV169 plus RV170, several signals, the largest and most prominent of which was approximately 2.7 kb, were obtained in the reaction with DNA of infected NIH 3T3 cells. These were absent in the other samples. For PCR with primers RV169 plus RV151, signals were present in the reactions with DNA from infected NIH 3T3 cells and GP+envAM12. A positive signal was also obtained with plasmid pCRII-c11, which bears the cloned PCR-amplified fragment obtained from the reaction shown in panel b.
FIG. 4
FIG. 4
Nucleotide sequences at recombination sites in helper virus. (a) Recombination site between 4070A env and MoMLV sequences derived from the pBabeNeo vector. The sequences shown here span the 3′ end of env, the downstream untranslated segment, and the 5′ end of the 3′ LTR. The following sequences are depicted: MoMLV (in the pBabeNeo vector, the neo gene is linked to nontranslated MoMLV sequences 3′ of env), the final 12 coding nucleotides of 4070A env, Friend MLV (in the packaging construct penvAm, present in GP+envAM12 cells, the 3′ end of 4070A env is linked downstream to a sequence derived from Friend MLV), and the sequence of the helper virus. Comparisons of these sequences suggest that a recombination event has occurred between the 3′ end of 4070A env and MoMLV sequences in the untranslated region (from pBabeNeo), as indicated by the arrow. The bases in the Friend MLV sequence which are printed in boldface type and underlined indicate differences compared with the helper virus sequence, suggesting that the Friend MLV sequence does not contribute to the genome of the helper virus. (b) Recombination between endogenous polytropic sequences (which contribute to MCF-type viruses) and 4070A amphotropic virus. Nucleotides 5638 to 5696 of the 5′ LTR–gag-pol PCR-amplified fragment (from Fig. 3b), corresponding to the 3′ portion of pol, are shown and compared with the corresponding sequences from the MCF-type virus pRFM#6 (this represents one of several MCF-type sequences in the database to which the helper virus bears >99% homology at this location) and the 4070A amphotropic retrovirus. The nucleotides which differ between the helper virus and either of these two other viral sequences are underlined and printed in boldface type. The shaded box represents a possible location where recombination between the two viral sequences to form the helper virus sequence may have occurred.
FIG. 5
FIG. 5
Structure of the helper virus genome as suggested by the PCR and RT-PCR studies. The virus arose by recombination events between endogenous retroviral sequences, the transcomplementing packaging construct penvAm, and the pBabeNeo vector, which is derived from MoMLV.
FIG. 6
FIG. 6
Amplification of a fragment linking endogenous polytropic retroviral sequences and 4070A env. Primer HC4 identifies with the endogenous polytropic retroviral sequence, while RV170 lies in 4070A env. A signal was obtained with DNA from infected NIH 3T3 cells. No amplification was achieved in reactions with DNA from parental NIH 3T3 cells, GP+envAM12 cells, and an early stock of GP+envAM12/pBabeNeo producer cells. As a positive control, plasmid pCRII-c3 was used, which contains the cloned PCR-amplified fragment linking the endogenous ecotropic sequence to 4070A env, as obtained from the reaction in Fig. 3c.
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