Effect of a viral rep gene on transformation of cells by an adeno-associated virus vector

VIROLOGY

166,6 12-6 15 (1988)

Effect of a Viral rep Gene on Transformation

of Ceils by an Adeno-Associated

Virus Vector

ELLA MENDELSON,’ MERWN G. SMITH,~ IRVING L. MILLER, AND BARRIE 1. CARTER Laboratory

of Molecular and Cellular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, Building 8, Room 304. National Institutes of Health, Bethesda, Maryland 20892 Received March 3 1, 1988; accepted June 22, 1988

Adeno-associated virus (AAV) vectors readily express the gene for geneticin-resistance under control of the AAV pm promoter when chromosomally integrated at low copy number in mammalian cells. We show that a truncated AAV rep gene, transcribed from the pS and plS promoters, mediates a negative effect on expression of geneticin-resistance in human 293 cells and a positive effect in HeLa cells. Also, we describe a novel phenotype for a mutant expressing the Q 1999Academic PWSS, I~C. pls rep gene alone which has a negative effect in 293 cells but no oositive effect in HeLa cells.

examine the effect of additional mutations in the rep gene of pJDT277 upon expression of rep protein and transformation to gen’in 293 and HeLa cells. The AAV genome map, the four AAV rep proteins, rep78, rep68, rep52, and rep40 (6, 74) and the structures of several AAV-neo vectors are shown in Fig. 1. pJDT277 contains a deletion of the AAV capsid gene and an insertion of a neo gene coding sequence at the HindIll site under control of the AAV p40 promoter (10). pMGSl20 contains a rep gene identical to pJDT277 but has a cis-dominant ori- defect and cannot be complemented for replication (7). pMGS127 (ori-, rep-) can neither replicate nor be complemented for AAV replication (7 I). pTMK5 and pTM20 contain frame-shift mutations in the rep gene and are both rep- for AAV DNA replication (data not shown). To examine the rep gene products, each vector was transfected into adenovirus-infected 293 cells and cytoplasmic and nuclear protein extracts were prepared and analyzed using the immunoblotting technique (6). The rabbit antibody used was raised against the rep protein produced in Escherichia co/i by a bacterial expression vector (74). The four wild-type AAV rep proteins, rep78, rep68, rep52, and rep40, extracted from cells infected with adenovirus and AAV, are shown in Fig. 2. In cells transfected with pJDT277 or pMGSl20 the cytoplasm contained a 68-kDa rep protein from the p5 transcription unit and a 40-kDa rep protein from the p,g transcription unit whereas in the nucleus, the 68kDa protein was predominant. Transfection with pMGSl27 yielded no rep proteins. pTMK5 yielded only the 40-kDa protein from the mRNA in the cytoplasm. pTM20 produced a 56-kDa protein (from the p5 RNA) that was more abundant in the nucleus than in the cytoplasm and a 28-kDa protein (from the pig RNA) in the nucleus (Fig. 2). The distribution of rep proteins

We have used the entire AAV genome cloned into a pBR322-derived plasmid pAV2 (5) to construct AAV vectors using the viral pgOpromoter to express foreign genes (70, 12). AAV vectors containing the prokaryotic chloramphenicol acetyltransferase (CAT) gene gave high levels of transient expression when transfected into human 293 (adenovirus-transformed human embryonic kidney) cells or HeLa cells in the absence of helper virus (7 7, 72). The viral rep gene mediated a net rrans-acting negative regulatory effect on CAT expression in 293 cells but a net positive effect in HeLa cells (7 7). The positive regulatory effect was also observed in adenovirus-infected human KB and HeLa cells (4, 7 7) and probably reflects increased transcription from AAV promoters (4, 73). The negative regulation by rep is not specific for AAV transcription promoters or RNA structure (3, 73) and may reflect translational level inhibition (13). These trans-regulatory effects of rep are independent of its function in AAV DNA replication (7, 9). We also used an AAV vector pJDT277 (Fig. 1) to express, from the p40 promoter, the prokaryotic neo (neomycin phosphotransferase) gene which mediates resistance to the antibiotic geneticin @en3 in mammalian cells. When pJDT277 was introduced into human cells by DNA transfection or as a viral transducing vector it integrated at low copy number and gen’was stably expressed (10). pJDT277 (Fig. 1) contains a rep gene which is truncated but nevertheless is functional in allowing replication in adenovirus-infected HeLa or 293 cells and rescue from the cellular genome (70). We did not determine if this truncated rep gene effected neo expression from the AAV p40 promoter. In this study we

’ To whom requests for reprints should be addressed. 2 Present address: Department of Biochemistry, University Otago, P.O. Box 56, Dunedin. New Zealand. 0042-6822/88

$3.00

Copyright Q 1989 by Academic Press, Inc. All nghts of reproduction in any form resewed

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612

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L-l @a

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-

ps 0

- 3.3Kb

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10

pm \2u

s

30

B

P40 \40

b r:i;2,

Bs HKl

2.6Kb

3.9Kb - 4.2Kb 50

60

70

80

90

I

I

I

I’?!

K2

100 Ba

Kl pJDT277 pMGS120

h t+

pMGS127

pTMK5

pTM20

FIG. 1. The structure of AAV vectors. The AAV genome (4675 nucleotides) (8) is shown on a scale of 100 map units. Restriction sites are indicated: Ba, Ball; S, Sstl; B, BarnHI; Bs, BstEll; H, HindIll; Kl, K2, Kpnl. Solid boxes, terminal repeat sequences comprising the replication origins; solid circles, transcription promoters p5, pls and phO; caret, intron; arrowhead, poly(A) site. Cytoplasmic RNAs are indicated at the top by horizontal arrows. Major open reading frames (on) are shown by rectangular boxes. For the 2.3-kb mRNA, on-2 coding for the major capsid protein VP3 is indicated (hatched box) but the reading frames for the minor capsid proteins VP2 and VP1 are omitted. For or-f-1 (open boxes) the locations of reading frames for rep78 (4.2-kb mRNA), rep68 (3.9-kb mRNA), rep52 (3.6-kb mRNA), and rep40 (3.3-kb mRNA) are shown. pJDT277 was derived from pAV2 by deletion of the Kpnl fragment and insertion of the neo coding sequence at the HindIll site (5, 10). The plasmid-derived portions of the vector are omitted for clarity. pMGSl27 (ori-, rep-) and pMGSl20 (ori-, rep’) were derived from pJDT277 by insertion of the 5stEIIIKpnl neo fragment from pJDT277 into the corresponding sites of pTS127 (11) or pPSl2 (7) respectively. pTMK5 was generated by cleaving pJDT277 with Sstl, removal of the 4-base overhanging end with DNA polymerase I Klenow fragment and religation. pTM20 was constructed by cutting pJDT277 with BstEll, removal of the 5-base overhang with Mung bean exonuclease, and religation.

between nucleus and cytoplasm was generally similar to that of wild-type (6, 14) or an analogous CAT construct (14) in that the p5 rep products were found predominantly in the nuclei, and the p,g products were found predominantly in cytoplasmic fractions except in pTM20 where a p,g product was found only in the nucleus. Thus, the appropriate truncated rep proteins were produced by each vector as predicted from its structure. The 68-kDa p5 product and the 40-kDa p,g product expressed by pJDT277 and pMGS120 were truncated at the carboxyl-terminus by termination codons in the leader sequence of the neo gene similarly to the products of AAV fep+vectors containing the CAT gene inserted at the HindIll site (14). A predicted 18-to 20-kDa protein from the p5 transcription unit in pTMK5 was not detected because it was too small or unstable. The frame-shift mutation in pTM20 (Figure 1) truncates rep immediately downstream of the BstEll site at a termination codon found in a non-rep reading frame. The

613

rep proteins shown in Fig. 2 were also detected after transfection of uninfected 293 cells and adenovirus-infected or uninfected HeLa cells, (data not shown). The frans-regulatory functions of the rep proteins of the AAV-neo vectors were examined in transient expression assays in 293 and HeLa cells by cotransfecting an indicator rep- AAV CAT vector, pTS6 1, expressing CAT from the p40 promoter (1 I), and the AAV-neo constructs (Table 1). In 293 cells, the rep+ constructs pJDT277 and pMGSl20 inhibited expression of CAT from the p40 promoter of pTS61 as compared to the rep- constructs pMGS127, pTM20, and pTMK5. Thus, expression of both p5 and pig products from pJDT277 or pMGSl20 had a net negative effect on expression from p40 in 293 cells whereas expression of pig product alone from pTMK5 or the truncated p5 and pig proteins from pTM20 had no effect. In HeLa cells there was an activation of four- to sixfold in CAT expression from pTS6I when both p5 and pig products were supplied by cotransfecting with either pJDT277 or pMGSl20 but little or no effect of the p,g product alone (pTMK5) or the truncated p5 and p,g products (pTM20). Expression of the neo gene was examined by transfecting each of the AAV-neo vectors into 293 cells or

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CR

C

N

C NC?+N

‘Rep78 Rep68 Rep52 sReo40

CR

FIG. 2. rep proteins expressed from AAV-neo constructs. Adenovirus-infected 293 cells were transfected with 10 pg of the plasmid DNAs indicated at the top. Protein extracts from nucleus (N) and cytoplasm (C)were prepared 48 hr later and analyzed by Western blotting with an anti-rep antiserum (14). All procedures were as described (6). The left-hand tracks (Ad) show extracts from Ad-infected 293 cells transfected with control pBR322 DNA. The right lane shows a whole cell extract from 293 cells coinfected with adenovirus and AAV, and the four viral rep proteins are indicated at the right. The location of rep protein products of the p6 or p19 transcription units of the AAV-neo constructs are indicated by arrowheads or open circles, respectively. CR indicates cross-reacting cellular proteins of 75 kDa in the nucleus and 30 to 38 kDa in the cytoplasm.

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614 TABLE 1

EFFECT OF TRUNCATED rep GENE PRODUCTS IN COTRANSFECTIONS WITH A rep- AAV-CAT VECTOR’ HeLa cells6

293 ceW Cotransfecting AAV-neo vector

CAT activity”

Normalized”

CAT activity0

pJDT277 pMGSl20 pMGS127 pTM20 pTMK5

5.0 5.3 14.7 14.7 13.8

0.34 0.36 1 .o 1.3 0.94

7.1 4.4 1.2 1.9 1.6

Normalized” 5.9 3.7 1 .o 1.58 1.33

a 293 or HeLa cells (1 06cells/35-mm dish) were cotransfected with a rep- AAV-CAT vector, pTS6 1, and an AAV-neo vector using the CaPO, procedure and 48 hr after transfection extracts (150 ~1)were prepared and assayed for CAT activity (11). pTS61 is analogous to pJDT277 but has a CAT gene inserted at the Hindlll site and a 750nucleotide deletion in the rep gene between the &/II and BsfEll sites (6, 17) and produces no rep gene products. ’ 293 cells were transfected with 1 pg each of pTS61 and the cotransfecting plasmid and HeLa cells were transfected with 2 rg of each plasmid. The total amount of DNA in each transfection mixture was adjusted to 10 pg with carrier plasmid DNA. c CAT activity is expressed as the percentage of the [Y]chloramphenicol substrate which was acetylated by 10 pl of 293 cell extract or 40 ~1of HeLa cell extract in 1 hr at 37”. d For each cell line CAT activity was normalized to that present in the contransfection with pMGS127 which cannot express any rep protein.

HeLa cells and growing in selective medium containing geneticin. The frequencies of gen’ colonies obtained with each vector in a series of experiments are summa-

rized in Table 2. The absolute gen’ frequency varied in each series of experiments, probably reflecting variations in cell growth and transfection efficiency (IO), but a consistent series of observations emerged. In 293 cells (Table 2) the rep-vectors pMGS127 and pTM20 showed an increased frequency of gen’ colonies compared to either rep+ vector, pJDT277 or pMGSl20. These results imply a negative effect of rep upon cellular transformation to gen’and are consistent with the transient assays in 293 cells (Table 1). In contrast, the rep- vector pTMK5 behaved similarly to the rep+ vector pJDT277 in the gen’ transformation assay (Table 2). This differs from the behavior of pTMK5 in the transient complementation assay (Table 1) and suggests that the pig rep protein produced by pTMK5 (Fig. 2) may be able to inhibit cellular transformation to gen’ in 293 cells. In HeLa cells (Table 2) the rep+ vectors (pJDT277, pMGS120) were three- to eightfold more efficient in gen’ transformation than the rep- vectors (pMGS127, pTM20, pTMK5). These results were consistent with those from the transient assays (Table 1). Thus, in HeLa cells the presence of both the p,g and the p5 rep gene products from pJDT277 or pMGS120 was associated with a significant increase in the frequency of gen’ colonies compared to no rep at all (pMGS127), the p,g product alone (pTMK5), or the truncated products of pTM20. The rep gene in pJDT277 or pMGSl20 is truncated at the HindIll site (nucleotide 1882). It produced mutant rep proteins that functioned for AAV DNA replication (10) and, as shown here, mediated negative or positive

TABLE 2 TRANSFORMATION

OF CELLS WITH AAV-neo VECTORS~

Frequency (x 1OW4)of gen’ colonie$ 293 cells Vector

1

pJDT277 pMGS120 pMGS127 pTM20 pTMK5

24 2 2 (7) 20 f 2 (7) 243 t 26 (5)

HeLa cells

2 7.6t4(4)

19.8 f 1 (4) 9.4 zk 1 (4)

3

1

8.3 f 3 (7)

24 3~2 (4) 56 iz 2 (4) 7 f 0.5 (4)

31 8

+6(5) k 3 (5)

2 10.4*

1

(.1)

2.2 + 0.4 (4) 1.2 + 0.4 (4)

a 10’ HeLa or 293 cells growing in 35-mm dishes at 37” in Eagle’s minimum essential medium supplemented with 10% fetal calf serum were transfected with 10 pg (series 1) or 5 pg (series 2, 3) of vector DNA. Forty-eight hours later the cells were ttypsinized and replated at varying numbers from 1 O4 to 10’ cells per 8-cm dish. Forty-eight hours later (ie., 4 days after transfection) geneticin (G418, GIBCO Laboratories) was added at 500 pg/ml of active component for 293 cells and 600 pg/ml active component for HeLa cells. The cultures were maintained in the selective medium for 14-20 days then gen’colonies were counted. Colonies were fixed and stained with 0.1% (w/v) crystal violet in 0.1 M citric acid for 1 hr and washed with water prior to counting. b In each series of experiments the frequency of gen’transformation (number of gen’ colonies per 1 O4 cells) was calculated for each vector. The number of determinations for each vector is shown in parentheses.

615

SHORT COMMUNICATIONS

regulation of CAT expression in a transient assay or gen’ expression in the cell transformation assay. In contrast, truncation of the rep gene at the BstEll site (nucleotide 1700) as in pTM20, or deletion of rep as in pMGS127, inhibited all three functions of rep. Thus, the expression of the gen’ marker was correlated with a functional rep gene in the same cell-specific way as in transient assays (1 I). These results also imply that the region of the rep gene downstream of the HindIll site is not absolutely required whereas the 61 amino acid region between the BstEll (nucleotide 1700) and the /-findIll (nucleotide 1880) sites is critical. The function of this latter region is not simply for rep protein stability because both pTM20 and pJDT277 synthesized amounts similar to wild-type virus. pTMK5, which allowed synthesis of the truncated p,g rep protein but not the p5 protein did not replicate and showed neither positive nor negative regulation of CAT expression in the transient assays. This is consistent with previous evidence that the p,g rep protein alone cannot mediate replication or trans-regulation (1, 4, 11). In the gen’ assay on HeLa cells, pTMK5 behaved similarly to pTM20 and in the same fashion as in the transient assay. In contrast, in 293 cells PTMK5 appeared to mediate negative regulation on gen’ expression in the cell transformation assay even though it did not do so in the transient assay. This conclusion agrees with experiments by Labow et a/. (3) reported after our work was done. These workers (3) used an AAV genome containing a mutation, ins1 1, in the p5 rep gene which should not have affected production of p,g rep proteins although they did not directly assay rep protein. Nevertheless, the ins1 1 mutant showed a trans-inhibitory effect on gen’ expression in a line of mouse melanoma cells. These observations provide strong evidence for a phenotype ascribable to the p,g rep protein alone which is only clearly demonstrated in the transformation assay using the gen’ marker. This is significant because it is the only property known for the p,g rep protein. The basis for this phenotype remains to be determined. Perhaps after integration at low copy number

in 293 cells, the p,g rep gene alone may mediate the negative effect whereas at high copy number in transient assays the p5 rep gene alone or together with the plgrep gene may be required. Alternatively, at low copy number the p5 gene product may be replaced by a cellular factor. These models are particularly interesting in view of the recent observations (73) that the negative regulation mechanism is post-transcriptional, apparently affecting the translation of the p40 messages. This, together with the presence of p,g protein products primarily in the cytoplasmic fraction, correlates with the major role that the p,g product may play in the negative regulation. A third model is that the p,g rep gene has a negative effect on growth of some cells, such as 293 cells, which may be independent of any regulation of expression from p40 and thus would not be observed in transient assays.

REFERENCES

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