Sep 15, 1983 - Shank, P. R., and M. Linial. 1980. Avian oncovirus mu- tant (SE2161b) deficient in genomic RNA: characteriza- tion of a deletion in the provirus.
MOLECULAR AND CELLULAR BIOLoGY, Dec. 1983, p. 2180-2190 0270-7306/83/122180-11$02.00/0 Copyright © 1983, American Society for Microbiology
Vol. 3, No. 12
Retrovirus Transduction: Generation of Infectious Retroviruses Expressing Dominant and Selectable Genes Is Associated with In Vivo Recombination and Deletion Events ALEXANDRA L. JOYNER AND ALAN BERNSTEIN* Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M4X IK9 Received 13 April 1983/Accepted 15 September 1983
We describe the generation of infectious retroviruses containing foreign genes by an in vivo recombination-deletion mechanism. Cotransfection into mouse cells of chimeric plasmids carrying a murine retrovirus 5' long terminal repeat and either the thymidine kinase (tk) gene of herpesvirus or the dominant selectable bacterial gene for neomycin resistance (neo), along with a clone of Moloney murine leukemia virus, results in the generation of infectious thymidine kinase or neomycin-resistant viruses. Expression of the selectable marker in these viruses can be regulated by the homologous transcriptional promoter of the gene, by the promoter contained within the Friend spleen focus-forming virus long terminal repeat, or by the simian virus 40 early region promoter. In all cases, the rescued viruses appeared to arise by recombination in vivo with Moloney murine leukemia virus sequences, resulting in the acquisition of the Moloney 3' long terminal repeat and variable amounts of the 3' adjacent Moloney genome. In two of the thymidine kinase constructs where tk was inserted 200 base pairs downstream from the long terminal repeat, the rescued viruses acquired a large part of the murine leukemia virus genome, including the region involved in packaging genomic RNA into virions. The generation of infectious neomycin-resistant virus is associated with deletions of simian virus 40 splicing and polyadenylation sequences. These results demonstrate that nonhomologous recombination and deletion events can take place in animal cells, resulting in the acquisition or removal of cis-acting sequences required for, or inhibitory to, retrovirus infectivity.
Studies on the life cycle and genomic structure of retroviruses have indicated that these viruses are naturally occurring transducing agents. The genomes of acutely transforming retroviruses include transforming or v-onc sequences that have been acquired from homologous cellular c-onc sequences during the replication of a nontransforming or slowly transforming retrovirus. Current models suggest that a series of integration, deletion, transcription, and recombination events are required to generate transmissible retroviruses containing these novel transforming genes (30). The acquisition of potential cellular transforming genes by acute retroviruses or by the experimental insertion of foreign sequences into retroviruses by recombinant DNA techniques (14, 18a, 21, 24, 28, 33) is usually accompanied by the deletion of viral sequences that encode replicative functions. Although these viruses are replication defective, they can be rescued as infectious virus after providing the missing functions in trans by coinfection with a replication-
competent helper virus. Retrovirus genomes also contain a number of regulatory regions that are essential for viral DNA synthesis, integration, transcription, and packaging of new genomic RNAs into nascent virions (for a review, see reference 30). These regulatory regions, located within and adjacent to the long terminal repeats (LTRs) present at the termini of an integrated provirus, act only in cis and cannot be provided for by a helper virus. In addition to these cisacting sequences, there may also be sequences which inhibit virus replication. For example, removal of the 3' end of the herpesvirus thymidine kinase gene (tk) has been observed to increase the yields of an infectious spleen necrosis virus vector containing an inserted tk fragment (21). In an attempt to elucidate some of the events that may take place during the generation of novel transforming retroviruses and to explore the versatility of retroviruses as gene transfer vectors, we constructed a series of recombinant DNA clones containing a 5' murine retrovirus
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VOL. 3, 1983
LTR and either the herpesvirus tk gene or the bacterial gene for neomycin resistance (neo). This latter gene is a dominant selectable marker in animal cells which confers resistance to the antibiotic G418 (26). All of the clones are deleted of 3' sequences necessary in cis for virus replication and integration, two clones contain the tk gene inserted within the region implicated in the preferential packaging of viral genomes into virions, and one construct contains simian virus 40 (SV40) polyadenylation and splicing sequences. Nevertheless, we show here that (i) infectious retroviruses expressing the tk or neo gene are generated after cotransfection with Moloney murine leukemia virus (MLV) DNA, (ii) these viruses appear to arise in vivo by recombination between the LTR-TK or LTRNEO constructs and the Moloney helper virus, (iii) two infectious TK retroviruses derived from clones containing the tk gene inserted 200 base pairs (bp) downstream from the 5' LTR appear to have acquired sequences from the MLV packaging region, and (iv) the generation of NEO viruses is associated with the deletion in vivo of SV40 polyadenylation and splicing regions. MATERIALS AND METHODS Cells and virus infections. The recipient LTA cell line used in the DNA-mediated gene transfer experiments is an adenine phosphoribosyl transferase-negative (APRT-) derivative of TK- mouse L cells (16) derived by R. Hughes and P. Plagemann. The Rat-2 cell line used to determine the titers of the TK and NEO viruses is a TK- derivative of Rat-1 cells (29) derived by W. Topp (Cold Spring Harbor Laboratory) and obtained from J. Hassell (McGill University). The NIH 3T3 cells (12) used for transfection and rescue of the recombinant viruses were obtained from G. Cooper (Harvard University). All cell lines were maintained in a-minimal essential medium (27) supplemented with 10%o fetal calf serum. Infection of Rat-2 cells plated 12 h previously at 2 x 104 cells per 60-mm plate was carried out in 10 ,ug of polybrene per ml for 18 h at 37°C. For selection of TK+ colonies, the cultures were switched to HAT (0.1 mM hypoxanthine, 1.0 ,uM aminopterin, 40 ,uM thymidine) medium 24 h later, and TK+ colonies were scored after 7 days of HAT selection. For selection of G418-resistant transductants, cells were passaged to 100-mm plates in the presence of G418 (400 ,g/ml; generously provided by Schering Corp.) 20 h after infection, and G418-resistant colonies were scored after 7 days of selection. Construction of recombinant plasmids. (i) TK vectors. The construction of recombinant plasmids pAJ11, pAJ12, pAJ13, and pAJ23 containing the Friend spleen focus-forming virus (SFFV) 5' LTR, adjacent viral sequences, and the herpesvirus tk gene has been described (15). The important features of these clones are indicated in Table 1. (ii) NEO vectors. The plasmid pLTR.SV2neo contains the bacterial neo gene under SV40 control, the Moloney 5' LTR, and 2.7 kilobases (kb) of 5' Moloney MLV sequences (see Fig. 5) cloned into a pBR322derived plasmid. pLTR.SV2neo was constructed by
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ligating equal amounts (1 ±Lg) of EcoRI-BamHI-digested pSV2neo (26), kindly provided by P. Southern (Scripps Research Foundation) and obtained from M. Buchwald (University of Toronto), with a gel-purified, 5.5-kb EcoRI-BamHI fragment corresponding to the 5' virus cell-junction fragment of an integrated MLV provirus. This latter fragment was obtained by preparative gel electrophoresis and electroelution (5) from the molecular clone pMOV-3 (10), kindly provided by R. Jaenisch (Heinrich-Pette-Institut, Hamburg, FRG). All plasmids were transfected into Escherichia coli HB101 (3). Plasmid DNA was prepared from 5 ml of bacterial cell suspension by the rapid boiling method (11). Large-scale plasmid preparations were purified by CsCl-ethidium bromide density gradient centrifugation. DNA transfection and virus rescue. LTA cells (7 x 10W) in 100-mm petri dishes or 2 x 105 NIH 3T3 cells in 60-mm petri dishes were transfected with plasmid DNA mixed with LTA or Rat-2 carrier DNA as described previously (9) with modifications (34). LTA cultures were switched to HAT medium 40 h later, and TK+ colonies were scored after 14 days. NIH 3T3 cells cotransfected with recombinant LTR-TK or LTR-NEO plasmids and pMOV-3 helper DNA were passaged twice, 2 and 5 days after cotransfection, and virus was harvested 3 to 4 days later. Blot hybridization and gel electrophoresis. Phage T4 DNA ligase (New England Biolabs) and bacterial alkaline phosphatase (Bethesda Research Laboratories) were used as described previously (15). Restriction endonuclease digestions were carried out with 2 to 4 U of enzyme per ,ug of DNA for 12 h under the conditions specified by the supplier (Boehringer Mannheim or Bethesda Research Laboratories). For DNA transfer and hybridization, 20 ,ug of each digested DNA was ethanol precipitated, suspended in buffer (4% Ficoll, 0.012 M EDTA, 0.5% sodium dodecyl sulfate, 0.01% bromophenol blue), and run on a 4.5mm vertical 1% agarose gel. Gels were treated and transferred to nitrocellulose filters (BA85; Schleicher & Schuell Co.) as described by Southern (25). Filters were prehybridized at 42°C for 12 h in 50%o formamide, Sx SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5 x Denhardt solution, 200 ,ug of sonicated salmon testes DNA (preheated to 100°C for 5 min) per ml, and 0.02 M sodium phosphate (pH 6.5) and hybridized for 18 h at 42°C in the same solution containing 10% dextran sulfate and 32P-labeled DNA (1 x 106 to 2 x 106 cpm/ml) essentially as described by Wahl et al. (31). High-specific-activity [32P]DNA probes (1 x 108 to 2 x 108 cpm/Ipg of DNA) were prepared by using a nick-translation kit (Bethesda Research Laboratories). After hybridization, the filters were washed twice for 5 min each at room temperature and twice for 20 min each at 50°C with 2x SSC and 0.1% sodium dodecyl sulfate, followed by two washes for 30 min each at 50C in 0.1x SSC and 0.1% sodium dodecyl sulfate. Filters were air dried and exposed to Kodak XAR-5 film with Cronex Lighting-Plus intensifying screens at
-700C. RESULTS Rescue of infectious TK virus. Plasmids containing the herpes simplex virus tk gene inserted downstream from the Friend SFFV LTR have
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MOL. CELL. BIOL.
TABLE 1. Generation of infectious TK virus Virus titefr TK'
Primary
CFU/ml Secondary
1
0
d
pYY508
0
0
pAJ11
1.53
1-10
>102 (clone 11-lA)
0.92
1-10
>103 (clones 12-1B, 12-1A,
Plasmid
Schematic of vector'
Relative TK+ transformation
efficiencyb Bomvui
lvuB Bam
pXI Sstl Bgll
pAJ12
TR
Bam
--K
12-3A)
pAJ23
1.07
103 (clones 23-2B, 23-1A)
L >102 (clone 13-1A)
