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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 Replication-Defective Chimeric Helper and Factors Affecting Generation of Competent : Expression of Moloney Murine Leukemia Virus Structural 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 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 . 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 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 (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 Bacterial and viral and cellular DNAs. 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 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 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 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 (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. MTMLVenv is the chimeric helper provirus designed to express the MoMuLV env function via the mMT-I promoter. MLVtkNEOI and MLVtkNEOII are proviral vectors designed to transduce G418 resistance. The light uneven line represents herpes simplex virus type 1 thymidine kinase promoter sequences. The heavy uneven line represents sequences encoding the Tn5 neomycin resistance gene. Straight lines represent viral sequences. These two proviral vectors differ in the viral sequences bordering the inserted tkNEO sequence. (B) Detailed comparison of the 5' ends of MoMuLV provirus, chimeric helper proviruses, and G418r-transducing proviral vectors described in panel A. * is a cis-acting sequence needed for efficient packaging of viral RNA into virions (27). Regions of the MoMuLV LTR indicated include the inverted repeats (IR), the terminal repeats of the RNA genome (R), and sequences unique to the 5' (U5) and 3' (U3) ends of the RNA genome. The hatched regions of the mMT-I promoter confer metal regulation (46). The solid region of the mMT-I sequences includes the 5' noncoding region of the first exon. 1800 BOSSELMAN ET AL. MOL. CELL. BIOL.

pMTMLVgagpol helper proviruses were assembled. MTMLVgagpol was de- pMTMLVenv signed to express only the gag-pol genes of the MoMuLV pNEO provirus, and MTMLVenv was designed to express the env TRANSFECT is truncated at the NIH-3T3 CELLS gene. The former putative splice acceptor AND for the gag-pol mRNA, and the latter is truncated at the SELECT G418'r XbaI site located 10 base pairs upstream of the env initiator M CELL CLONES ATG. All viral sequences upstream of these positions were deleted and replaced by a 1.8-kilobase EcoRI-to-BgiII frag- G41 8r ment of mouse DNA, which includes the mMT-I promoter. The helper DNA designed to express gag-pol also contains the env gene sequence. However, since no known splice SCREEN FOR RT PRODUCTION AND ISOLATE RT+ CLONES donor sequence is present within the mMT-I promoter fragment that was used in this construct, the second helper DNA was designed specifically for env expression. RT+ G418r Each defective helper provirus was positioned between the EcoRI and HindIII sites of plasmid pSVO8. The gene organization of the original MoMuLV provirus and the J/ |CTEST FOR COMPETENT VIRUS BY RT , XC ASSAY derivative chimeric helper proviruses are shown in Fig. 2. of chimeric helper proviruses into NIH 3T3 VIRUS- cells and isolation of virus-negative helper cell line clone 32. CLONE 32 RT- The chimeric helper proviruses were cotransfected into NIH Br G41 1/ 3T3 cells with After 10 to 14 of |I/ ISOLATE CLONES HARBORING A along plasmid pNEO. days SELECTABLE VECTOR BY G418 selection, cells of individual G418' clones were trans- l/ TRANSFECTION WITH MLVtkNEO I ferred to 24-well The cells were for several AND pSV2gpt FOLLOWED BY plates. grown MYCOPHENOLIC ACID SELECTION passages and analyzed for release of RT (16). About 10% of the clones analyzed released RT activity, presumably due to CLONE 32 MPAI assembly of RNA-negative virions (40, 53). +MLVtkNE I G41 8' All RT-positive clones tested were negative in the XC plaque assay (XC-) (37), indicating that env gene expression TEST FOR TRANSDUCTION OF was insufficient to cause syncytia formation and that wild- G418r TO NIH-3T3 CELLS TEST FOR REPLICATION type ecotropic virus was not being released. Clone 32 was COMPETENT VIRUS chosen for more detailed study because it released the highest levels of RT activity. CLONE 32-5A YIELDS 2x104 G41 8rCFU/mI Southern blot analysis of clone 32 DNA confirmed the MASS CULTURE OF CLONE 32 + MLVtkNEO I (154 Independent Transformants) presence of both transfected helper DNAs (43) (data not YIELDS 104 G418' CFU/mI shown). Analysis of the spent growth medium showed a two- BOTH CLONE 32-5A AND THE MASS CULTURE to fivefold increase in RT production in cultures containing ARE VIRUS NEGATIVE 100 ,uM ZnSO4, indicating that expression of viral gene FIG. 3. Diagram of procedures used to derive and characterize products was under control of the mMT-I promoter (8, 28). ecotropic helper cell lines. MPA, Mycophenolic acid. Furthermore, the titer of ecotropic MoMuLV was reduced 30-fold on clone 32 as compared with that on NIH 3T3 cells, indicating presence of the env gene product on the cell linkers were prepared by the method of Caruthers (5). surface (38). The relative contribution of the two helper Enzymes used to modify DNA were used in accordance with DNAs to total env expression is not known. Early passages specifications of the manufacturer. of clone 32 did not release XC+ virus and did not transduce RT assay. Reverse transcriptase (RT) was assayed by the RT activity to RT-negative NIH 3T3 cells (data not shown). procedure described by Goff et al. (16). Samples (25 ,ul) of Generation of the clone 32 helper cell line is diagrammed in medium were mixed with 50 .1l of reaction cocktail and Fig. 3. incubated at 37°C for 60 min. Samples were analyzed by Assembly and sequence organization of vector proviruses. autoradiography or in a Beckman scintillation counter. Two different approaches were used in the construction of the vector proviruses whose assembly is diagrammed in Fig. 4. Viral sequences containing portions of the structural RESULTS genes of the virus were removed by restriction enzyme digestion and replaced by sequences encoding TnS neomycin Construction of replication-defective chimeric helper provi- phosphotransferase and the herpes simplex virus type 1 ruses. The helper proviruses described here were designed to thymidine kinase promoter. The substituted sequences in be defective for certain cis-acting functions needed for MLVtkNEOI were positioned between the PvuI site at replication and nondefective for trans-acting viral functions nucleotide 423 and the ClaI site at nucleotide 7676 from the needed for virion assembly. These replication-defective normal 5' cap position of the genomic MoMuLV RNA. helper proviruses are chimeric and were derived from the A second vector, MLVtkNEOII, carrying the same select- mMT-I promoter (9) and structural genes from the provirus able marker was constructed by deleting a smaller region of of MoMuLV (1, 41). The 5' end of the MoMuLV provirus the MoMuLV proviral genome extending from the BglII site was removed and replaced by the mMT-I promoter. This at nucleotide 1907 to the HpaI site at nucleotide 7198 from results in the loss of numerous cis-acting functions necessary the 5' end of the genomic MoMuLV RNA. The deleted viral for viral replication (13, 14, 18, 41). The assembly of these sequences in both constructs include regions encoding por- helper proviruses is shown in Fig. 1. Two of these defective tions of the gag gene, all of the pol gene, and a large portion VOL. 7, 1987 EXPRESSION OF MoMuLV GENES VIA mMT-I PROMOTER 1X()l

pMLV26 ( PvuI or HpaI ) pNEO at 423 -pSVO8 pBR322 ul or HpaI BglI CpaalCHII { amHI NruI Pvul

-

3 56 EcoRI I Clal BamHI Hpal NruI KLENOW

FORMER X BamHI Nrul

EcoRI BgIlI Hpal i\ NruI pMLVtkN EO I

1LIGASE

pMLVtkNEO II pSVO8 FORMER FORMER BglllBamHl NrulHpal Clal Hill

EcoRI

FIG. 4. Schematic diagram showing assembly of G418r-transducing vectors MLVtkNEOI and MLVtkNEOII. , Plasmid sequences;

C1, LTRs; X -i, viral sequences; c, herpes simplex virus type 1 sequences; , sequences encoding the Tn5 neomycin phosphotrans- ferase gene; , cellular flanking sequences; HIII, HindIll.

of the env gene. These two vector DNAs are shown in Fig. competent virus. Clone 32 cells should provide all the tratl.s- 2. acting viral functions necessary for assembly of infectious Significant differences exist between MLVtkNEOI and replication-defective vectors. Plasmids pMLVtkNEOI and MLVtkNEOII at their 5' ends in relation to the helper pSV2gpt (34) were cotransfected into clone 32 cells to proviruses already described. RNA transcripts from determine whether this cell line could assemble infectious MLVtkNEOI and the helper proviruses share no overlap- replication-defective vector. A total of 164 clones were ping sequence homology along the entire 5' region of their selected in methotrexate and mycophenolic acid (34). The . In contrast, RNA transcripts from vector levels of G418r CFU produced by some of these clones are MLVtkNEOII have a 1,343-nucleotide overlap with RNA shown in Table 1. A pool of 154 of these clones released an transcripts from the helper provirus MTMLVgagpol. average titer of 104 G418r CFU/ml. This pool of vector- Clone 32 supported assembly of replication-defective infec- producing clone 32 cells remained XC- and failed to trans- tious vectors without generation of detectable replication- duce RT activity even after 2 months in culture. As a test for 1802 BOSSELMAN ET AL. MOL. CELL. BIOL.

TABLE 1. Release of G418F CFU by clone 32 cells transfected tional cis-acting functions from the 3' end of the with MLVtkNEOIa MTMLVgagpol helper DNA might prevent this type of No. of G4181 recombination. Clone CFU/ml, assayed Comparison of clone 32 with helper cell lines Psi-2 and on NIH 3T3 cells PA317. Mass cultures of the Psi-2 cell line or clone 32 each

32-1A ...... 0 carrying MLVtkNEOI were established by transfection and 32-2A ...... 2.3 x 103 growth in selective medium. G418r-transducing vectors from 32-SA ...... 2.6 x 104 these cultures were then used to establish separate G418r 32-6A ...... 0 populations of NIH 3T3 cells, each representing >200 inde- 32-1B ...... 0 pendent clones. These two populations of MLVtkNEOI- 32-3B ...... 2.5 x 102 infected NIH 3T3 cells were then analyzed for release of 324B ...... 1.2 x 104 G418r CFU on NIH 3T3 cells. G418r cultures made by Mass culture of 154 clones ...... 1.0 X 104 infection with Psi-2-derived vectors transduced an average a Individual clones or mass cultures of clone 32 stably transfected with of 1.2 x 102 G418r CFU/ml to NIH 3T3 cells in two pMLVtkNEOI and pSV2gpt were assayed for release of G418r CFU. The independent tests. However, these second-generation cul- titers in medium harvested after 6 h of incubation were determined by tures of G418r NIH 3T3 cells did not contain detectable infecting NIH 3T3 cells, counting the surviving colonies present in culture after selection in 400 p.g ofG418 per ml, and multiplying by the dilution factor. replication-competent viruses and did not transduce G418r by subsequent infection of NIH 3T3 cells. This has also been observed with the amphotropic helper cell line Psi-AM, wild-type virus, 1 ml of filtered medium from these pooled which is similar in derivation to the Psi-2 ecotropic helper clones was placed on NIH 3T3 cells, which were then passed cell line (7). Secondary passage of G418r from populations of in culture continuously for 30 days. These cultures remained G418r cells infected with Psi-2-derived vectors may repre- XC-, did not express RT, and did not transmit G418 sent leakiness of the packaging-defective Psi-2 genomes. In resistance to fresh cultures of NIH 3T3 cells (Fig. 5). contrast, G418r NIH 3T3 cells established by infection with Titers produced by six individual isolates of helper cell MLVtkNEOI derived from clone 32 cells did not transduce clone 32 cotransfected with pMLVtkNEOI and pSV2gpt G418r. These results are shown in Fig. 6. ranged from 0 to 2.6 x 104 G418r CFU/ml (Table 1). As a test We also tested the helper cell line PA317 for the ability to for wild-type virus, an individual clone (32-5A) of helper cell support assembly of the vector N2 without generating com- clone 32 harboring MLVtkNEOI and releasing 2.6 x 104 petent virus, as reported by Miller and Buttimore (29). The G418r CFU/ml was expanded to about 3 x 108 cells. Filtered N2 vector shares overlapping sequence homology with the culture fluid (1 ml) was passed onto NIH 3T3 cells. After 24 PA317 helper DNA. PA317 was transfected with N2 DNA days in culture, these cells were XC-, did not express containing the G418r gene. Drug-resistant clones were detectable RT, and did not pass G418 resistance to fresh pooled and assayed for virus. Mass cultures were negative NIH 3T3 cells by infection. for virus after three passages, as judged by the absence of Differences in the sequence structure of defective proviruses RT production in NIH 3T3 cells cultured for 25 days after in clone 32 affected the generation of recombinant replication- infection. However, after nine passages the same mass competent virus. Vector DNAs MLVtkNEOI and culture of PA317 carrying the N2 vector was virus positive MLVtkNEOII were transfected independently along with by the same criterion. These results underscore the impor- pSV2gpt into the clone 32 cell line to determine how differ- tance both of multiple cis-acting defects in the helper DNA ences in vector sequence structure and organization affected and of the absence of overlapping homologous sequences generation of replication-competent virus. between helper and vector genomes (32, 51). Clone 32 cell lines transfected with MLVtkNEOII were Because the reported titers of some vectors produced by rescued by selection in mycophenolic acid and methotrex- Psi-2 and PA317 cells are higher than those we observed, ate. Some of these clones were XC+, indicating the presence clone 32, Psi-2, and PA317 cells were compared directly for of replication-competent ecotropic MuLV. The XC- clones the ability to assemble the vector MLVtkNEOI. Psi-2 and were carried in culture and assayed for the appearance of clone 32 cells were transfected with vector DNA. PA317 infectious ecotropic virus by XC assay. Over a period of 1 to cells were infected with vector produced by clone 32. 2 weeks, all of these cultures became XC+ and exhibited Drug-resistant cultures were assayed for transduction of elevated levels of RT, indicating that the presence of G418r to NIH 3T3 cells. All of these helper cells were MLVtkNEOII and its RNA transcripts in clone 32 leads to comparable with respect to assembly of the vector generation of ecotropic MuLV (data not shown). In contrast, MLVtkNEOI. Titers from mass cultures were 1 x 104 virus could not be detected in these cells transfected with CFU/ml for clone 32, 3 x 10" CFU/ml for Psi-2, and an MLVtkNEOI, even after extended culture. average of 1.4 x 104 CFU/ml for 10 PA317 clones. A factor Clone 32 became XC+ and released replication-competent which may have contributed to the difference in titers virus after long-term culture in vitro. After about 4 months in between the vector used here and others is that continuous culture, clone 32 began to release infectious MLVtkNEOI lacks 145 base pairs of the previously defined virus. Cultures of clone 32 cells which produced this virus MoMuLV packaging signal (27) and an even more extensive had not been transfected with a rescuable vector. Virus- (>1 kilobase) region of the 5' viral gag gene present in the positive cultures became XC+. Furthermore, medium from N2 vector (11, 29). these clone 32 cultures could transduce this property to uninfected NIH 3T3 cells. The virus produced in these DISCUSSION cultures probably derived from recombination between helper provirus and endogenous cellular sequences capable Design and construction of chimeric helper proviruses. of restoring viral functions needed for replication. The Portions of the viral genome deleted from the chimeric precise origin of this recombinant and the events involved in helper DNAs encode a number of cis-acting viral functions its generation have not been investigated. Deletion of addi- and include the inverted repeat implicated in integration of VOL. 7, 1987 EXPRESSION OF MoMuLV GENES VIA mMT-I PROMOTER 1803

MASS CULTURED CLONE 32 + MLVtkNEO I

GROW 1.5x107 CELLS FROM A POOL OF 154 CLONES OF LINE 32 + MLVtkNEO I (PASSAGE 12)

I CULTURE FLUID

TRANSFER FILTERED CULTURE FLUID TO 5x1 05 NIH-3T3 CELLS IN MEDIA CONTAINING 8 ug/mI POLYBRENE

PASSAGE CELLS FOR 30 DAYS

ASSAY FOR ASSAY FOR XC+ COCULTIVATE 7 DAYS REVERSE TRANSCRIPTASE: VIRUS: WITH NONPRODUCER NIH-3T3 CELLS CARRYING NEGATIVE NEGATIVE THE DEFECTIVE PROVIRUS MLVtkNEO I

ASSAY FILTERED CULTURE FLUID FOR TRANSDUCTION OF G41 8r TO NIH-3T3 CELLS: NEGATIVE FIG. 5. Diagram of procedure used to detect competent virus in mass cultures of clone 32 harboring MLVtkNEOI. 1804 BOSSELMAN ET AL. MOL. CELL. BIOL.

-1 .5x 107 Clone 32+MLVtkNEOI -1 .5x104r2+MLVtkNEOI detected, even after 2 months of culture. These results and those for individual clones are shown in Fig. 5 and Table 1. Infect NIH-3T3 Cells Infect NIH-3T3 Cells However, transfection of clone 32 cells with the vector MLVtkNEOII resulted in very frequent release of replica- Pool 249 G418r Clones Pool >1.000 G418' Clones tion-competent virus in addition to G418r-transducing vec- Expand Culture to Expand Culture to tor. MLVtkNEOII and the helper provirus MTMLVgagpol -1.5x107 Cells -1.5x107 Cells share 1,343 nucleotides of homologous sequence overlap at their 5' ends. Since this is the major difference between Harvest Culture Fluid and Harvest Culture Fluid and Infect 5s105 NIH-3T3 Cells Infect 5x 1 05 NIH-3T3 Cells vectors MLVtkNEOI and MLVtkNEOII, homologous over- lapping sequences probably play a major role in promoting Select in G418 Select in G418 recombination to form replication-competent virus. The helper proviruses resident within clone 32 cells are an No Surviving Colonies 397 Surviving Colonies obvious substrate for this process, but other endogenous sequences present within the genome may also be involved. Although we have not observed release of competent virus from clone 32 cells harboring MLVtkNEOI, we have twice observed the appearance of virus in cultures of clone 32 itself. This suggests that endogenous sequences are available which can provide functions necessary for virus formation. The appearance of virus in clone 32 cells was rare relative to the occurrence of virus in clone 32 harboring MLVtkNEOII, ~~~* O0 suggesting that the events involved in generation of virus in these two situations are different. These observations also \* indicate that even deletion of multiple cis-acting functions from the helper provirus may not always be sufficient to \ .* prevent leading to formation of com- V *. petent virus. Comparison with other packaging-defective retroviral genomes and helper cell/vector systems. Cell lines which FIG. 6. Comparison of clone 32 and Psi-2 cells with respect to harbor packaging-defective helper proviruses derived from transmission of both vector and helper proviruses. Medium from ecotropic MoMuLV (27), the amphotropic MuLV 4070A (7, cultures of each helper cell line harboring MLVtkNEOI was used to 31, 42), and avian reticuloendotheliosis virus strain A (51) infect NIH 3T3 cells. G4181 clones were pooled and assayed for subsequent transmission of the vector MLVtkNEOI by infection have been reported to support the assembly of infectious and selection. The culture dishes contained G4181 colonies of NIH vectors in the absence of detectable replication-competent 3T3 cells derived by infection with medium from cultures of cells virus. Deletion of the cis-acting packaging sequence was infected with the vector MLVtkNEOI generated by either clone 32 sufficient in some instances to generate genetically stable (left) or Psi-2 (right) cells. A total of 397 G418T CFU were detected helper cell lines (7, 27, 30), whereas in another instance a in culture fluid from NIH 3T3 cells infected with Psi-2-derived second cis-acting defect in the helper provirus was necessary MLVtkNEOI. The plate shown represents 1/10 of the surviving to prevent generation of replication-competent virus by colonies. No detectable G4181 CFU were detected in cultures recombination between the helper and vector sequences infected with clone 32-derived MLVtkNEOI. (42). Deletion of the cis-acting packaging sequence from a murine retroviral vector decreased assembly of the vector the provirus and located at the ends of the 5' long terminal RNAs into virions by 1,000-fold, from about 106 to 103 IU/ml repeat (LTR) (36), the transcriptional control sequences (26). This assembly of packaging-defective viral RNA into located in the U3 region of the LTR including the enhancer virions is similar to viral packaging of the cellular globin sequences (21, 25), the CAT and TATA boxes (41), and the mRNA in Friend virus-infected hematopoietic cells (19). R sequence which may facilitate the jump between the 5' and These observations are consistent with the apparent 3' ends of the RNA genome during the process of reverse transduction of the packaging-defective Psi-2 genome which transcription (14). Extending beyond the 3' boundary of U5, we observed (Fig. 6). Since high-frequency recombination these deletions also include the tRNA primer-binding site for among retroviruses (44, 45, 50, 54) seems to be dependent initiation of viral minus strand DNA synthesis (18) and splice upon formation of heterozygous virions (52, 55, 56), these donor and splice acceptor sequences which may be used in results indicate that deletion of the packaging sequence the formation of subgenomic gag-pol and env mRNAs (41). alone may not be sufficient to prevent such defective RNAs The deleted region also includes the sequence which falls from participating in genetic recombination. In fact, low- between the splice donor and acceptor for the gag-pol level recombination between resident genomes of a naturally transcript, a cis-acting sequence which has been shown to be occurring packaging mutant of Schmidt-Ruppin Rous sar- necessary for efficient assembly of viral genomic RNA into coma virus type A and fully competent Rous-associated virions (27). This region of the MoMuLV provirus also virus-2 has been demonstrated after extended in vitro culture includes a nonessential sequence necessary for expression of (22, 24). a glycosylated form of the gag polyprotein, gPr80gag (10, 39). In contrast, the chimeric helper proviruses described here Clone 32 supported assembly of infectious vectors, but were not transduced along with the vectors they were vector sequence structure affected generation of competent designed to complement. Thus, the use of such mutiply virus. A pool of 154 clones of clone 32 cotransfected with defective helper proviruses eliminates the slow spread of pMLVtkNEOI and pSV2gpt produced about 104 G418r packaging-defective and secondary transduction CFU/ml. No detectable replication-competent virus could be of vector sequences in target cell populations. VOL. 7, 1987 EXPRESSION OF MoMuLV GENES VIA mMT-I PROMOTER 1805

Recombination between related viruses is probably based 6. Colbere-Garapin, F., F. Horodniceanu, P. Kourilsky, and A.-C. largely on sequence homology and may involve reverse Garapin. 1981. A new dominant hybrid selective marker for transcription of heterologous RNAs copackaged into higher eukaryotic cells. J. Mol. Biol. 150:1-14. virions. This can be reduced by 7. Cone, R. D., and R. C. Mulligan. 1984. High-efficiency gene transfer into mammalian cells: generation of helper-free recom- using helper proviruses and vectors whose RNAs share binant with broad mammalian host range. Proc. Natl. overlapping deletions in nonessential regions of the genome Acad. Sci. USA 81:6349-6353. adjacent to the 3' end of the packaging sequence. This 8. Durnam, D. M., and R. D. Palmiter. 1981. Transcriptional strategy reduces generation of wild-type virus in the regulation ofthe mouse metallothionein-I gene by heavy metals. reticuloendotheliosis virus vector system (51) and may con- J. Biol. Chem. 256:5712-5716. tribute to the genetic stability reported for the murine 9. Durnam, D. M., F. Perrin, F. Gannon, and R. D. Pahmiter. 1980. ecotropic and amphotropic helper cell systems previously Isolation and characterization of the mouse metallothionein-I described (7, 30, 42). However, together the helper and gene. Proc. Natl. Acad. Sci. USA 77:6511-6515. 10. Edwards, S. A., and H. Fan. 1981. Immunoselection and char- vector genomes still retain all essential viral functions and acterization of Moloney murine leukemia virus-infected cell could generate competent virus by an illegitimate recombi- lines deficient in surface gag antigen expression. nation. Detailed studies of Moloney murine sarcoma virus 113:95-108. (47, 48) and Moloney mink cell focus-forming virus (2) 11. Eglitis, M. A., P. Kantoff, E. Gilboa, and W. F. Anderson. 1985. suggest that recombination can involve both cellular and Gene expression in mice after high efficiency retroviral- viral information and that extensive sequence mediated gene transfer. Science 230:1395-1398. homology is not an absolute prerequisite for formation of 12. Feinberg, A. P., and B. Vogelstein. 1983. A technique for . Although nonhomologous recombination radiolabeling DNA restriction endonuclease fragments to high is not well understood, it has been shown that unrelated specific activity. Anal. Biochem. 132:6-13. DNAs transfected into L cells recombine close to regions of 13. Gilboa, E., S. Goff, A. Shields, F. Yoshimura, S. Mitra, and D. Baltimore. 1979. In vitro synthesis of a 9 kbp terminally redun- partial sequence homology (3). In light of these findings, the dant DNA carrying the infectivity of Moloney murine leukemia lack of both sequence homology and overlap between the 5' virus. Cell 16:863-874. ends of the chimeric helper and vector genomes described 14. Gilboa, E., S. W. Mitra, S. Goff, and D. Baltimore. 1979. A here should minimize generation of virus by recombination detailed model of reverse transcription and tests of crucial between the two and with the endogenous viral sequences of aspects. Cell 18:93-100. the host genome. Another approach to the design of retro- 15. Glanvifle, N., D. M. Durnam, and R. D. Palmiter. 1981. Struc- viral helper DNAs has been described, involving substitu- ture of mouse metallothionein-I gene and its mRNA. Nature tion of the 3' LTR with a simian virus 40 polyadenylation (London) 292:267-269. signal (29). However, the vector N2 used in this study 16. Goff, S., P. Traktman, and D. Baltimore. 1981. Isolation and properties of Moloney murine leukemia virus mutants: use of a contained homologous sequence overlaps with the helper rapid essay for release of virion reverse transcriptase. J. 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Fan. 1984. fusion plasmids injected into mouse eggs. Nature (London) Nonfunction of a Moloney murine leukaemia virus regulatory 296:39-42. sequence in F9 embryonal carcinoma cells. Nature (London) 5. Caruthers, M. H. 1982. Chemical synthesis ofoligodeoxynucleo- 308:470-472. tides using the phosphite triester intermediates, p. 71-79. In H. 26. Mann, R., and D. Baltimore. 1985. Varying the position of a G. Gussen and A. Lang (ed.), Chemical and enzymatic synthe- retrovirus packaging sequence results in the encapsidation of sis of gene fragments: a laboratory manual. Verlag Chemie, both unspliced and spliced RNAs. J. Virol. 54:401-407. Weinheim, Federal Republic of Germany. 27. Mann, R., R. C. Mulligan, and D. Baltinore. 1983. Construction 1806 BOSSELMAN ET AL. MOL. CELL. BIOL.

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