APOBEC3 proteins are packaged into retrovirus virions and can hypermutate retroviruses during reverse transcription. We found that HT-1080 human fibrosarcoma cells hypermutate retroviruses, and that the HT-1080 cell-derived FLYA13 retrovirus packaging cells also hypermutate a retrovirus vector produced using these cells. We found no hypermutation of the same vector produced by the mouse cell-derived packaging line PT67 or by human 293 cells transfected with the vector and retrovirus packaging plasmids. We expect that avoidance of vector hypermutation will be particularly important for vectors used in gene therapy, wherein mutant proteins might stimulate deleterious immune responses.
Retroviruses are subject to mutagenesis by APOBEC3 proteins present in host cells. These proteins are packaged into virions and cause frequent G to A base changes in the retroviral sense strand by deaminating cytidines on the antisense DNA strand during virus reverse transcription.1 Retroviral vectors are also subject to such hypermutation when produced in cells that overexpress APOBEC3 proteins, but there appear to be no studies of mutagenesis of vectors made for use in cell culture or for gene therapy. In the case of gene therapy, such mutagenesis could result in production of multiple variants of the intended therapeutic protein that are inactive and/or stimulate immune responses.
Typically, retroviral vectors are made by transient transfection of 293 human embryonic kidney cells with the vector and with expression plasmids encoding the required viral proteins, or by vector transfer into retrovirus packaging cell lines that constitutively produce the retroviral proteins required for vector production.2 APOBEC3 levels vary among different cell types, but the activity of APOBEC3 proteins in cells used to make retroviral vectors has not been studied. Here, we show that vectors produced by packaging cells made using HT-1080 human fibrosarcoma cells, but not vectors produced by packaging cells made using NIH 3T3 fibroblasts or by transient transfection of 293 cells, show dramatic G to A hypermutation characteristic of APOBEC3 modification.
Results and discussion
Evidence that APOBEC3 mutagenesis might affect vectors made from cells used to make retrovirus packaging cell lines came from an experiment in which we sequenced a recombinant vector produced from HT-1080 cells infected with the LAPSN vector and xenotropic murine leukemia virus-related virus (XMRV).3 The LAPSN vector encodes alkaline phosphatase and bacterial neomycin phosphotransferase (neo),4 and the recombinant vector (LNras*SN; GenBank accession GU934326), consisted of the LAPSN vector with an insertion of the Nras oncogene from the HT-1080 cells in place of AP sequences. Interestingly, vector sequencing revealed multiple G to A mutations in the neo coding region retained in the recombinant vector, indicative of APOBEC3-mediated mutagenesis (Figure 1; LNRas*SN). One of the mutations introduced a stop codon that inactivated the neo gene by truncating the neo protein after amino acid 14.
To test whether hypermutation of the recombinant vector was simply a rare event, we used virus from HT-1080 cells infected with the LAPSN vector and XMRV (HT-1080/LAPSN+XMRV cells) to infect 208F rat fibroblasts, PCR-amplified the neo coding regions from the integrated copies of the LAPSN vector, and sequenced 10 plasmid clones made from the PCR product. Note that no selection for vector expression was performed before PCR amplification. Four of the ten clones showed significant (P⩽0.03) G to A hypermutation (Figure 1), indicating a high rate of hypermutation by APOBEC3.
To test whether vectors made by retrovirus packaging cells derived from HT-1080 cells might also be mutated by APOBEC3 proteins, we used virus from HT-1080-based FLYA13 packaging cells5 transduced with the LAPSN vector (FLYA13/LAPSN cells) to infect 208F rat fibroblasts, PCR-amplified the neo coding regions from the integrated copies of the LAPSN vector, and sequenced 20 plasmid clones made from the PCR product. Sequencing revealed G to A hypermutation in 5 of the 20 clones (Figure 1), showing that these packaging cells do indeed produce hypermutated vectors at a significant level, and that hypermutation of vectors made from HT-1080 cells does not require the presence of a replicating retrovirus.
Packaging cells derived from NIH 3T3 cells and 293 cells are widely used to make retrovirus vectors, thus we tested for hypermutation of the LAPSN vector produced by these cells. Virus from NIH 3T3-based PT67 cells6 transduced with the LAPSN vector (PT67/LAPSN cells) was used to infect 208F rat cells and the neo coding regions of 20 integrants were determined, revealing only one or no G to A mutations in each of the 20 clones (Figure 1). Similarly, virus produced by transient transfection of 293 cells with the LAPSN vector and retrovirus packaging plasmids pLGPS (Gag-Pol)7 and pSX2 (10A1 amphotropic virus Env)6 was used to infect 208F rat cells, and sequencing of 20 integrants revealed no hypermutation (Figure 1). Thus, we did not detect APOBEC3-mediated hypermutation in vectors produced by PT67 or 293 cells.
In summary, our results demonstrate frequent G to A hypermutation of retroviral vectors produced from HT-1080 human fibrosarcoma cells and from the HT-1080-based FLYA13 (amphotropic host range)5 retrovirus packaging cells. We expect hypermutation of vectors produced by other HT-1080-based packaging cells, including the FLYRD18 (RD114 tropic),5 HX (xenotropic) (ATCC CRL-12011) and HP (polytropic) (ATCC CRL-12012) cell lines. HT-1080 cells make relatively high levels of APOBEC3B and 3C mRNAs,8 and presumably the encoded proteins are responsible for hypermutation of virus made by HT-1080 cells. Such hypermutation is also expected from packaging cells made using the CEM human T-cell line, which expresses a high level of APOBEC3G (CEM15) protein,9 and was used to make the CEMFLYA packaging cells.10
We did not detect hypermutation of a retroviral vector by the NIH 3T3 mouse cell-derived PT67 packaging cell line. The lack of hypermutation is consistent with previous findings that mouse cells express only one APOBEC3 gene (in contrast to human cells which can express seven different APOBEC3 genes), murine leukemia viruses are relatively resistant to inhibition by the mouse APOBEC3 protein, and this inhibition apparently does not involve hypermutation.11, 12, 13, 14 Thus, we predict no hypermutation of vectors made by other mouse cell-derived packaging cell lines, especially those made using the same NIH 3T3 cells and the same Moloney murine leukemia virus gag-pol gene used to make PT67 cells, such as PA317 (amphotropic)15 and PG13 (gibbon ape leukemia virus tropism)7 packaging cells. However, we were surprised to find random mutations in the neo coding regions of ∼50% of the integrated viruses produced by virus from PT67/LAPSN cells. This rate of mutation (19 mutations in 795 × 20 bases=1.2 × 10−3) is higher than expected for the high-fidelity Taq polymerase used to amplify the sequences (estimated by the manufacturer to be 2 × 10−6), and thus is likely due to errors during reverse transcription. The PT67/LAPSN cells were not cloned after transduction with LAPSN virus, thus the mutation rate is the result of errors accumulated in two rounds of reverse transcription. Indeed, the error rate observed for virus produced by transfection of 293 cells (10 mutations in 795 × 20 bases=6.3 × 10−4) represents errors from one round of reverse transcription, and is about half of the rate for the PT67/LAPSN virus, as would be predicted.
We did not detect hypermutation of vectors made using 293 cells (<5% of vector copies hypermutated), which is fortunate given their widespread use in the production of both retroviral and lentiviral vectors. However, interferon, and possibly other factors present in culture medium, can increase APOBEC3 levels in cells,16 and 293T cells, a derivative of 293 cells that express SV40 T-antigen and that have been used to make retroviral vectors, are reported to express APOBEC3C mRNA.8 Thus, it will be important to monitor for hypermutation of vectors made from 293 and other human cells to avoid vector mutagenesis that reduces vector titer and might stimulate immune responses in gene therapy recipients.
Materials and methods
Cell culture and viruses
A pseudodiploid subclone (HTX) of HT-1080 human fibrosarcoma cells (ATCC CCL-121) was used in all experiments. These cells, 208F rat fibroblasts,17 22Rv1 prostate carcinoma cells (ATCC CRL-2505), FLYA135 and PT676 amphotropic retrovirus packaging cells, PG13 gibbon ape leukemia virus-pseudotype retrovirus packaging cells7 and 293 human embryonic kidney cells18 were grown in Dulbecco's modified Eagle medium with 4.5 g l−1 glucose and 7% fetal bovine serum. PT67/LAPSN and PG13/LAPSN cells were made by transduction of PT67 and PG13 cells with LAPSN vector produced from PE501 NIH 3T3-cell-derived ecotropic retrovirus packaging cells19 transiently transfected with LAPSN plasmid, followed by growing the cells in G418 to select for cells expressing the LAPSN vector. FLYA13/LAPSN cells were made by transduction with LAPSN virus from PG13/LAPSN cells followed by selection of the cells in G418. LAPSN vector with an amphotropic pseudotype was generated from 293 cells by transient transfection of 293 cells with the pLAPSN,4 pLGPS7 and pSX26 plasmids. XMRV was obtained from 22Rv1 cells.20 HT-1080/LAPSN+XMRV cells were made by transducing HT-1080 cells with LAPSN vector produced by PT67/LAPSN cells, selecting the cells in G418, and infecting the cells with XMRV virus from 22Rv1 cells. All viruses were harvested from confluent dishes of cells fed 12–24 h previously, were filtered through 0.45 μm-pore-size filters, and were used immediately or were frozen at −70 °C for later use.
PCR amplification of neo sequences
The 208F cells were exposed to LAPSN viruses in medium containing 4 μg ml−1 Polybrene, and genomic DNA was collected after 24 h (DNeasy Blood and Tissue kit, Qiagen, Valencia, CA, USA). To avoid bias, selection for vector gene expression was not performed. PCR amplification of the neo sequence was done using Platinum Taq HiFi (Invitrogen, Carlsbad, CA, USA) with primers SNF2 (5′-CCTCTGAGCTATTCCA-3′) and SNR2 (5′-AAAATGGCGTTACTTAAG-3′). These primers were chosen to avoid neo coding strand GG dinucleotides and to minimize other potential sites of G to A activity that could bias the results by preventing amplification of mutated sequences. PCR products were gel extracted (QIAquick Gel Extraction Kit, Qiagen) and cloned into a plasmid (StrataClone PCR Cloning Kit, Stratagene, Santa Clara, CA, USA). Single clones were then isolated and sequenced using M13 primers. Sequences were analyzed using the Hypermut program (http://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html).
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We thank shared resources for DNA sequencing. This work was supported by National Institutes of Health grants UL1 DE19582, a Pilot grant from the Northwest Genome Engineering Consortium (to MJM and ADM); P30 DK47754, a Molecular Therapy Core Center grant (to ADM); and CA09229, a training grant (to MJM).
ADM is an inventor on several patents describing retrovirus packaging cell lines made using NIH 3T3 cells, including the PT67 cells used in this report. MJM declares no conflict of interest.
Supplementary Information accompanies the paper on Gene Therapy website
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Miller, A., Metzger, M. APOBEC3-mediated hypermutation of retroviral vectors produced from some retrovirus packaging cell lines. Gene Ther 18, 528–530 (2011). https://doi.org/10.1038/gt.2010.177
- retroviral vectors
- retrovirus packaging cells
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