Mutation Research, 220 (1989) 101-106 101 Elsevier

MTR02608

SV40-based shuttle viruses

C.F.M. Menck, C. Madzak *, G. Renault *, A. Margot * and A. Sarasin * Biology Department, Institute of Biosciences, USP CP 11461, S~o Paulo, 05499, SP (Brazil) and * Laboratory of Molecular Genetics,

Institut de Recherches Scientifiques sur le Cancer, CNRS, B.P. No. 8, 94802 Villejuif (France)

(Accepted 27 September 1988)

Keywords: Shuttle vector; SV40 encapsidation; DNA-induced alterations; DNA transfection; Viral infection

Summary

We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid D N A can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded D N A in bacteria. They allow the transfection of a plasmid genome into mammal ian cells, as either s in#e-stranded or double-stranded DNA.

Shuttle vectors replicate in both mammalian cells and bacteria. They provide an efficient method for studying mutagenesis induced during plasmid amplification in eukaryotic cells, by using the easy and well-known genetics of E. coli (Calos et al., 1983; Razzaque et al., 1983). We expect these vectors, while replicating in animal cells, to exhibit a chromatin structure similar to that de- scribed for animal viruses and the cellular ge- nome. Moreover, replication and repair processes occurring on the vector D N A are carried out only with cellular enzymes. Thus, alterations induced in these plasmids will reflect those occurring in the cell genome. This is reinforced by the observation that induced mutations in shuttle vectors are simi- lar, at the D N A sequence level, to those found in an endogenous gene (Drobetsky et al., 1988).

Correspondence: Dr. A. Sarasin, Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, CNRS, B.P. No. 8, 94802 Villejuif (France).

Several different shuttle vector systems are now available and are described in detail in this issue. In our approach, we concentrated our efforts on avoiding the introduction of vector into mam- malian cells by transfection of naked DNA. In- deed, D N A transfection is a chemical process that renders the cell membrane permeable to DNA, but not without interfering with the cell metabo- lism. This process is toxic to some cell lines and is inefficient, with usually less than 5% of the cells taking up a high number of D N A molecules (Wil- son, 1978). We developed simian virus 40 (SV40)- based shuttle vectors able to be packaged as pseudo-virions, which can then be propagated by infection (Fig. la). This special feature of the shuttle viruses allows their transmission to fresh cell cultures as a viral genome, so that D N A entering the cells is protected by the viral chro- matin and capsid structures.

We review here results on D N A replication, amplification and stability in monkey cells of some vectors we developed, comparing this novel system

0165-1110/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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filamentous pseudo-phages b) ~SVF1 system

. . / .,.,.

....... ! ......... ___ 1 ........ !~ Did~tzatio n :)ir ::ust;:tem~ bacteria / &

;As\ !)__.

infe~n ~ vlrl?

Fig. 1. Scheme of the shuttle virus system. (a) Classical shuttle virus: production of infectious particles, allowing the use of the vector for either infection or transfection of mammalian cells. (b) ~rSVF1 system: production of ss DNA of the vectors ~rSVF1-A or -B in bacteria, following infection with a helper phage. Three molecular forms of the vectors can be used for transfection in mammalian cells: ss DNA, duplex DNA and heteroduplex molecules, obtained by in vitro hybridization of ss ~rSVF1-A and -B.

wi th classical SV40 shut t le vectors. Moreover , a new series of vectors is desc r ibed that can be p r o d u c e d and in t roduced in the cells as single- s t r anded D N A (Fig. l b ) . Thei r fate and po ten t i a l use in s tudies involving the me tabo l i sm and muta - genesis of d a m a g e d s ingle-s t randed D N A are analyzed.

Vector replication and amplification in monkey cells

The bas ic s t ructure of shut t le viruses, previ- ous ly descr ibed b y M e n c k et al. (1987) is schema-

t ized in Fig. 2. These p lasmids car ry the SV40 or igin of D N A repl ica t ion and the late genes which code for the caps id prote ins . P lasmid func- t ions for rep l ica t ion and select ion in E. coli were p rov ided by the min ip l a smid ~rAplac (Seed, 1983) which was inser ted in to the ear ly SV40 genes, d i s rup t ing the large T ant igen gene. Since these p lasmids are unable to synthesize this prote in , they repl ica te only in permiss ive cells which pro- vide the T ant igen in trans, such as the m o n k e y COS7 cells (Gluzman , 1981). Fo l lowing transfec- t ion of COS7 cells wi th the p lasmid , infect ious par t ic les con ta in ing the vectors are re leased into the cul ture med ia (Menck et al., 1987). Virus stocks can therefore be p r o d u c e d and used to infect fresh cell cultures.

The a u t o r a d i o g r a p h y of a Southern b lo t (Fig. 3A) shows the rep l ica t ion of the vec tor ~rSV40AE2 (Fig. 2) in 3 m o n k e y cell l ines af ter viral infect ion. These cell l ines (COS7, CMT3 and BMT10) ex- pressed the large T ant igen gene at d i f ferent levels. COS7 cells p r o d u c e d this p ro te in cons t i tu t ive ly (Gluzman , 1981), while in the CMT3 and BMT10 cells the s t ruc tura l gene for the large T ant igen was under the cont ro l of the induc ib le mouse meta l lo th ione in p r o m o t e r (G e ra rd and Gluzman ,

Ceeorl] /

~ ~--[BsmHI] [Bol l ] "-[ B,~n HI] Bgl 1 Hind IIr

Bgl II

Fig. 2. General structure of shuttle viruses. The construction of the initial plasmids is described in details by Menck et at. (1987). Inner arrows show major transcripts for SV40 late genes (VP1, VP2 and VP3) and the bacterial suppressor tRNA (supF); lacO is the lactose operator sequence, and the DNA replication origins of SV40 (ori) and plasmid (~rori) are also indicated, y represents a variable region in each different vector: for ~rSV40A E2, the 5' part of the SV40 T antigen gene; for ¢rSVPC3, the trp promoter and cat gene; for ~rSVF1, the replication origin of ss bacteriophage fl.

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1985). The express ion of the T ant igen was s t rongly increased by the add i t i on of bo th zinc and c a d m i u m ions in the cell culture. W e fol lowed the accumula t ion of rep l ica t ing vectors with t ime af ter infec t ion in the 3 cell lines. As expected, repl i - ca t ion of shut t le viruses is grea t ly increased in C M T 3 and BMT10 cells by the add i t i on of meta l ions (Fig. 3A). This effect is obvious in CMT3 cells, where the a m o u n t of p lasmids in non - induced cells is the same as tha t found in COS7 cells. This a m o u n t is smal ler in BMT10 cells, due to a very low const i tu t ive synthesis of T antigen. In the i nduced state, the a m o u n t of vector is increased a p p r o x i m a t e l y 10-fold in CMT3 cells (Fig. 3A). In

COS7 cells, the add i t i on of meta l ions has no de tec tab le effect on ¢rSV40AE2 repl ica t ion . The a m o u n t of recovered p l a smid al lows the d i rec t v isual iza t ion of the D N A on an e th id ium- b romide - s t a ine d agarose gel (Fig. 3B). The vo lume loaded in each slot co r r e sponds to the f if t ieth pa r t of a D N A p r e p a r a t i o n f rom one conf luen t cell cu l ture dish (5 cm in d iameter ) . A s s u m i n g that 100% of the cells were infected, we es t imate that abou t 2 × 105 vector molecules pe r cell can be ob t a ined in COS7 cells. This n u m b e r increases to a p p r o x i m a t e l y 106 molecules pe r cell in C M T 3 cells dur ing the induc t ion phase. These values are much higher than those r epo r t ed for non- infec-

Fig. 3. DNA replication of ~-SV40 AE2 in monkey cell lines. Monkey cell lines, COS7, CMT3 and BMT10, were infected with ~rSV40AE2 virus and cells were cultivated in the presence (+) or absence ( - ) of zinc and cadmium (Gerard and Gluzman, 1985). Extrachromosomal DNA was extracted from cells (adapted for mammalian cells from Birnboim and Doly, 1979) harvested at the indicated times after infection. After agarose gel electrophoresis, DNA was analyzed by (A) Southern blotting using [32p]sv40 as a probe, or (B) directly by UV fluorescence of the ethidium bromide dye.

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tious shuttle vectors (see DuBridge and Calos, 1988).

This dramatic amphfication of plasmid DNA in monkey cells is probably due to the formation of virus particles, allowing the vectors to be trans- ferred from the infected cells to the rest of the cell population. With non-infectious SV40-based shut- tle vectors, the DNA molecules replicate only in the cells initially transfected and the maximum of DNA amplification is attained 2-3 days after transfection. The large amount of plasmid DNA obtained from a 5-cm culture dish facilitates the rescue of vectors back to E. coll. Several thousand bacterial colonies can be obtained from DNA extracted from only 1 dish of infected monkey cells.

Plasmid stability in mammalian cells

Numerous factors may interfere with the sta- bility of the shuttle viruses during their passage in mammalian cells. The size of the original plasmid is particularly important for its stability, due to limitations imposed by the SV40 capsid. We ob- served that plasmids with sizes greater than the SV40 genome (5.2 kb) were deleted to smaller molecules, probably due to selection during DNA encapsidation and propagation as virus. Inversely, plasmids smaller than 3.8 kb were often re- arranged to produce a larger genome which could be more efficiently packaged (Menck et al., (1988). Moreover, our results indicate that the maximum plasmid stability occurred when genome sizes were 4.0-4.8 kb. This stability seems to be due, at least in part, to the biological selection imposed by the viral packaging system for an optimal size.

In the first series of shuttle viruses, we ex- amined the plasmid stability using the 28-bp lac operator as a mutation target (Menck et al., 1987). In the bacterial host with multiple copies of plasmid carrying the wild-type lacO sequence, the lac repressor was titrated and the lac operon was derepressed. Under these conditions, the bacteria were able to metabolize the synthetic substrate Xgal (5-bromo-4-chloro-3- indolyl - f l -D-galac- topyranoside), rendering the colony blue. Modifi- cations in the plasmid lacO sequence, including point mutations or rearrangements, which did not

allow the lac repressor binding, yielded white or light blue colonies. We also constructed a new series of vectors (~rSVPC3) carrying the cat (chloramphenicol acetyltransferase) gene, under the control of the tryptophan promoter, facilitat- ing the manipulation in bacteria. The supF gene was used in these new vectors as a larger (85 bp) and more reliable mutation target. The screening of mutants in this gene was performed by the color assay described above, using the suppression of a lacZ amber mutation carried by an ap- propriate bacterial host.

In Table 1 we report the mutation frequency in some of our shuttle viruses under different condi- tions of treatment. Spontaneous mutagenesis levels are of the same order of magnitude as for other SV40-based shuttle vectors, except ~rSVPC3, for which it is lower (6 × 10-5). These values are low enough to allow the study of induced mutagenesis at the mutation loci. As expected, UV irradiation of 7rSV40AE2 and ~rSVPC3 virus leads to an increase in mutation frequency. There is no sig- nificant change in the mutation frequency when ~rSV40AE2 replicates in COS7 cells and in CMT3 cells in the presence of the metal ions. As dis- cussed above, the overexpression of large T anti- gen in these cells enhances the rate of replication of vector molecules (Fig. 3). Our results indicate that increasing the number of replicative cycles is without any visible effect on plasmid stability as measured by the lacO system.

Shuttle viruses that can be produced as single- stranded D N A

Single-stranded DNA (ss) is an important tool for studies concerning recombination and muta- genesis in mammalian cells. Several recombination models implicate ss intermediates (see Subramani, 1989). For example, the ss annealing model pro- posed by Lin et al. (1987) involves a strand-specific exonuclease that produces complementary ss re- gions. In mutagenesis, the use of ss DNA allows us to answer several questions such as: (a) the comparison of spontaneous or lesion-induced mutagenesis on ss or double-stranded (ds) mole- cules; (b) the study of the mutagenesis of hetero- duplex molecules carrying lesions in only 1 strand; and (c) the study of relations between replication

T A B L E 1

M U T A T I O N F R E Q U E N C Y IN S H U T T L E V I R U S E S

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G e n o m e a M u t a t i o n T rea tmen t W h i t e / b l u e M u t a t i o n (size) target colonies b f requency

• rSV40AE2 lacO none 1 0 / 1 4 7 0 3 7 X 10 -4 (4.0 kb) UV c = 1200 J / m 2 3 3 / 7 0 4 9 4.6 x 1 0 - 3

C M T 3 cells d 1 / 2 6 9 4 4 X 10 - 4

induced s ta te

~rSVPC3 supF none 5 / 8 1 3 9 9 6 X 10 -5

(4.6 kb) UV = 1000 J / m 2 3 / 2 2 0 4 1.3 x 10 -3

~rSVF1 e lacO ss D N A 8 / 2 7 9 3 7 3 x 10 -4

(4.6 kb) ds D N A 1 6 / 2 7 5 5 0 6 x 10 - 4

he te rodup lex 3 1 / 1 7 8 6 0 1.7 x 1 0 - 3

a The de ta i led s t ruc ture of each vector has been descr ibed (Menck et al., 1987) or is descr ibed in the text. A genera l s cheme is p re sen ted in Fig. 2.

b Mutagenes i s was de t e rmined as descr ibed in the text, us ing a w h i t e / b l u e color assay. Ex t r ach romosoma l D N A was ex t rac ted f rom

cells harves ted 7 days af ter infec t ion (for ~rSV40AE2 and ~rSVPC3) or t ransfec t ion (for ~rSVF1). c UV i r r ad ia t ion (254 rim) of virus before infec t ion of COS7 cells. d C M T 3 cells were infected wi th ~rSV40A E2 and meta l ions ( c a d m i u m and zinc) were added to the cul ture med ia as in Fig. 3. e The d i f ferent D N A forms were ob t a ined as descr ibed in the text.

and repair, in a system where lesions can be selectively introduced on the leading or the lag- ging strand.

We developed a shuttle virus system where the same D N A sequences can be used in 3 forms: ds, ss or heteroduplex containing 1 single-stranded eye (Madzak et al., 1989). The ¢rSVF1 vectors were constructed by inserting, in an SV40-based shuttle virus, an ss bacteriophage replication origin. The orientation (A or B) of this fragment (from the f l phage) determines which strand of the vector can be produced as a s s pseudophage in bacteria. Plasmids carrying the origin of f l phage enter in f l replication mode, in permissive bacteria, after infection with a helper phage. This generates ss plasmid D N A that is efficiently assembled as a pseudophage particle (Dotto et al., 1981; Dente et al., 1983). In our experiments, phage preparations were submitted to DNase I treatment, before D N A purification, to remove any contaminating DNA. The resulting ss D N A preparations were free of any detectable ds DNA. The ss D N A derived from ¢rSVF1-A and from ~rSVF1-B are comple- mentary to each other, except for the D N A frag- ment containing the f l ori. The in vitro hybridiza- tion of the 2 ss ¢rSVF1-A and -B leads to a

heteroduplex molecule with an ss eye correspond- ing to the fl ori fragment (Fig. lb).

Transfection experiments involving the 3 differ- ent forms of 7rSVF1 plasmids were performed in monkey COS7 cells. Cells were harvested 7 days after transfection and the extrachromosomal D N A analyzed. All the different forms of molecules, including ss and heteroduplex DNA, yielded ~rSVF1 duplex molecules (Madzak et al., 1989). Control experiments using S1 nuclease showed that our results were not due to a possible con- tamination of ds D N A in the ss D N A prepara- tion. Thus, ss 7rSVF1 can be converted into duplex D N A and replicate as such.

The spontaneous mutation frequency on the lacO target was determined for the 3 molecular forms of these vectors (Table 1). The ss molecules have the same level of stability in mammal ian cells as ds DNA. In contrast, the heteroduplex struc- ture exhibits a higher mutat ion frequency: the ss eye could induce mutations in sequences located at its borders, as is the case for lacO, in the heteroduplex molecules (Table 1).

Conclusion

The formation of infectious particles in cells infected with shuttle viruses provides a biological

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tool enabl ing the in t roduc t ion into m a m m a l i a n cells of plasmid D N A protected by the capsid proteins. Besides, the vector genome can be damaged in vitro without interfering with the cell viability. These vectors will be repaired, replicated and perhaps mutagenized by using the cellular enzyme machinery. Thus, the events which would result in changes in the D N A molecules are prob- ably similar to those that occur in cell chro- mosomes. The produc t ion by the infected cells of virus particles able to infect ne ighbor cells pro-

vides a very efficient method for p lasmid amplifi- ca t ion in m a m m a l i a n cells, facil i tat ing the re- covery in bacter ia and, consequently, the analysis of mutagenesis. In addit ion, the ~rSVF1 vectors

are interest ing tools to compare induced mutagen- esis on ss and ds molecules. The ease with which these vectors produce pure heteroduplex mole- cules may also permit the s tudy of D N A repli- ca t ion of templates conta in ing damage in only 1 strand. Therefore, the SV40-based shuttle viruses const i tute an al ternative system for answering m a n y specific quest ions on the molecular mecha- n isms of both mutagenesis and recombina t ion processes in m a m m a l i a n cells, con t r ibu t ing to their bet ter unders tanding .

Acknowledgements

C.F.M.M. is on the leave of the State Univer- sity of Rio de Janeiro and dur ing this work had a post-doctoral fellowship from C N P q (Brazil) and A R C (Villejuif, France). This work was supported by grants from ARC, from the F o n d a t i o n pour la

Recherche Mrdica le (Paris) and from the Com- mission of the European Communi t i e s (No. B16-

163 F, Brussels, Belgium).

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