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Replication-Defective Chimeric Helper Proviruses and Factors Affecting Generation of Competent Virus

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Replication-Defective Chimeric Helper Proviruses and Factors Affecting Generation of Competent Virus MOLECULAR AND CELLULAR BIOLOGY, May 1987, p. 1797-1806 Vol. 7, No. 5 0270-7306/87/051797-10$02.00/0 Copyright © 1987, American Society for Microbiology Replication-Defective Chimeric Helper Proviruses and Factors Affecting Generation of Competent Virus: Expression of Moloney Murine Leukemia Virus Structural Genes via the Metallothionein Promoter ROBERT A. BOSSELMAN,* ROU-YIN HSU, JOAN BRUSZEWSKI, SYLVIA HU, FRANK MARTIN, AND MARGERY NICOLSON Amgen, Thousand Oaks, California 91320 Received 15 October 1986/Accepted 28 January 1987 Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5' long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2 -inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 104 G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 x 104 CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system. Extensive and detailed information concerning the gene present data on the ability of a helper cell line to produce structure and organization of the Moloney murine leukemia infectious replication-defective vector and on factors which virus (MoMuLV) and the Moloney murine sarcoma virus affect the generation of replication-competent virus within (41, 48) has allowed generation of a number of vector this vector system. systems derived from these viruses (7, 27, 30, 31, 42). Among the vector systems which have been described, helper cells used to generate virus-free preparations of MATERIALS AND METHODS infectious replication-defective vectors may offer the great- Cells and Viruses. NIH 3T3 cells were grown in Dulbecco- est potential for both experimental and clinical applications Vogt modified Eagle medium containing 10% calf serum. XC (7, 27). cells were grown in Dulbecco-Vogt modified Eagle medium Helper proviruses carried by these cell lines have gener- containing 10o fetal calf serum. MoMuLV clone A was ally been derived from nondefective proviral DNAs by obtained from H. Fan, University of California at Irvine. deletion of a single cis-acting sequence needed for efficient Psi-2 cells were obtained with permission of R. Mulligan and assembly of viral RNA into virions (27, 31). The successful grown as described previously (27). PA317 cells were ob- use of helper cells and the vectors they produce will depend tained from the American Type Culture Collection, on systems which do not generate replication-competent Rockville, Md. virus. Because the biological properties of retroviruses Bacterial plasmids and viral and cellular DNAs. Plasmid which allow them to recombine with each other and their pSVO8 (35) was obtained from A. Bert with permission of R. hosts to form natural vectors may also contribute to the Tjian. Plasmid pMK (4) was obtained from R. Palmiter. generation of competent virus in these vector systems, we Clone 63 of the MoMuLV proviral DNA carried in the have taken a different approach to their design. recombinant lambda phage Charon 4A was obtained from H. This report describes a set of two chimeric helper provi- Fan (1). Plasmid pNEO was obtained from B. Wold and has ruses derived from MoMuLV and the biological character- been described elsewhere (V. C. Bond and B. Wold, sub- istics of a helper cell line derived by transfecting these mitted for publication). Plasmid pSV2gpt was developed by proviruses into NIH 3T3 cells. These chimeric helper provi- Mulligan and Berg (33) and was obtained from Bethesda ruses are defective for multiple cis-acting viral functions Research Laboratories, Inc. The N2 vector was obtained essential for viral replication and use the mouse metal- from E. Gilboa (11). lothionein I (mMT-I) promoter (9, 15) to express either the Virus infection. NIH 3T3 cells (5 x 105) were seeded in a gag-pol or env region of the original MoMuLV provirus. We 6-cm petri dish. Six hours later, freshly harvested culture fluid was filtered and applied to the cells in culture medium * Corresponding author. containing 8 ,ug of Polybrene per ml. Virus was allowed to 1797 1798 BOSSELMAN ET AL. MOL. CELL. BIOL. pMK pMLV5' IpMLV26 PstI pCFM 414 c0111^ Pstl X ) HII b Pstl Xhol Xbal Xbal Hi I I EcoRl 3 4 5 6 7 PARTIAL Pstl EcoRII Xhol Xhol pBR322 _ EcoRI BgIll Pstl Xhol coRl Xhol \t 1\ 2 3 4 5 6 7 BgIll-Pstl LINKER LIGASE PARTIAL Xbal pMTM LVgagpol EcoRl pBR322 CO I 911Pstl Xhol Hil 1 2 3 4 5 6 7 EcoRI BgIll (: oRI Xbal Xbal Hl 6I 7 67 BgIll-Hpal-Xbal LINKER rLIGASE pMTM LVenv pBR322 I Hpal Xbal Hll FIG. 1. Schematic diagram showing assembly of chimeric helper proviruses. pMK is a pBR322-derived plasmid containing a fusion gene composed of the mMT-I promoter (_) and the herpes simplex virus type 1 thymidine kinase coding sequence (VIED) (4). pMLV5' is a plasmid carrying the 5' region of an integrated MoMuLV provirus ( i) and adjacent cellular sequences (). pMLV26 is a plasmid carrying an entire integrated MoMuLV provirus together with flanking cellular sequences. , Plasmid sequences; O, LTR of the provirus. HIII, HindIII. adhere to the cells for about 16 h. After infection, the cells ferase gene carried in plasmid pSV2gpt, as described previ- were washed in phosphate-buffered saline, suspended by ously (34). trypsinization, and transferred to fresh plates at a dilution of DNA preparation, transfer, and analysis. Plasmid DNA 1:20. When virus titers were expected to be very low, newly was usually prepared from cultures of ampicillin-resistant infected cultures were transferred in total to 10-cm dishes on bacterial clones as described previously (20). DNAs were the first passage and thereafter diluted 1:20 on passage. introduced into NIH 3T3 cells by a modification of the XC assays. We used a modified version of the XC plaque calcium phosphate coprecipitation technique of Graham and assay for ecotropic MuLV (37). Stained cultures were ex- van der Eb (17). Plasmid DNAs were introduced into com- amined both microscopically for the formation of syncytia petent cells of Escherichia coli HB101 as previously de- and macroscopically for resulting plaques. scribed (49). Cloned isolates of bacteria which had taken up Selection. NIH 3T3 cells transfected with the TnS the DNA were selected on agar plates containing L broth neomycin phosphotransferase gene carried by plasmid and 80 ,ug of ampicillin per ml. The Southern blotting pNEO were selected in medium containing G418 at concen- technique was used to analyze cellular DNA for the presence trations ranging from 400 to 600 ,ug/ml (6). Cells were of viral sequences (43). Radioactive probes were prepared transfected with the xanthine-guanine phosphoribosyltrans- by the method of random priming (12). Synthetic DNA VOL. 7, 1987 EXPRESSION OF MoMuLV GENES VIA mMT-I PROMOTER 1799 Pvul 423 Ball PsU BgllI Xbw Hpal Clal A LTR 215 568 1907 5767 7198 7676 LTR MoMLV I ITG pol I. env I I SDI SAT TATG SD SA SA EcoRI gtill Clal mMT-1 PROMOTER Pstl Xbal I gag MTMLVgagpol --- I ATG pTITIIn I SA EcoRI E Clal mMT-L PROMOTER Xbal MTMLMVenv I J. env I ATG FORMER Pvul FORMER BPalR Cial g I ~HSVI tkPROMOTER NEO' |, MLVtk NEO I SD Pvul FORMER FORMER Bat! psti E I11 H CIal tkPROMOTER MLVtkNEOII I ga HSVI r . WaEI SD SA Pvul B Ball 423 PstI Bglll IR LTR IR 215 A68 /pol/env 1907 1 gag/ 0- MoMLV R U5 =~~~~~~~~~~~ - - - IU3ENHAKEROMERI I 1; ATG +1 SD SA EcoRI Bgill Pasl I A mMT-1 PROMOTER n n Inxlr /lnv MTMLVgagpoI L I,I TA-_A_ -= -1 750 -180 l-120 --0 +1 + A * EcoRI BglU Hpal Xbal MITMLVeni/L mMT-I PROMOTER ATG FORMER Ball Pvul LTR I I HSVI tkNEO MLVtkNEOI l I I I I L FORMER Bat PvuI Patl III LTR |HSVltkNEO, MLVtkNEO I I gag ,i r I I I + 1 SD SA FIG. 2. Comparison of parental MoMuLV provirus, chimeric helper proviruses, and proviral vectors. (A) The MoMuLV proviral genome (MoMLV) is shown at the top. The structural genes gag, pol, and env are shown as solid lines bounded by LTRs. Restriction endonuclease cleavage sites are numbered relative to the cap site of viral RNA, as described by Shinnick et al. (41). The relative positions of splice donors (SD), splice acceptors (SA), and initiator ATGs are given for the gag-pol and env RNA transcripts. MTMLVgagpol is the chimeric helper provirus designed to express the MoMuLV gag-pol function via the mMT-I promoter. The mMT-I promoter is shown at the left end of the construction (46). The broken line corresponds to viral sequences deleted from this construction.
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