JOURNAL
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Vol. 32, No. 2
VIROLOGY, Nov. 1979, p. 623-628
0022-538X/79/11-0623/06$02.00/0
Physical Mapping of a Large-Plaque Mutation of Adenovirus Type 2 G. CHINNADURAI,* SHANBAGAM CHINNADURAI, AND JOHN BRUSCA Institute for Molecular Virology, Saint Louis University School of Medicine, St. Louis, Missouri 63110 Received for publication 2 April 1979
We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 Sail-A fragment. By using this method, several recombinants were constructed between a large-plaque (Ip) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the Ip mutation is located within the left 41% of Ad2 genome.
The 31 recognized human adenovirus serotypes form five groups (A to E) based upon genomic DNA homologies in molecular hybridization experiments (M. Green, J. K. Mackey, W. S. M. Wold, and P. Rigden, Virology, in press). The DNA homologies of serotypes within each group range from 48 to 69% (group A) to 99 to 100% (group C). Because of this group-specific DNA homology, recombination between serotypes might be expected to occur during infections. Takemori (17) first demonstrated genetic recombination between cytocidal (cyt) mutants of adenovirus type 12 (Adl2) (group A). Subsequently, Williams and co-workers (19) demonstrated recombination among temperature-sensitive (ts) mutants of Ad5 (group C) and constructed a genetic map of the Ad5 genome based upon recombination frequencies. Although the genomes of Ad2 and Ad5 are similar, minor differences in base sequences exist because some restriction endonucleases generate different fragments from Ad2 and Ad5 DNA. These differences in cleavage patterns have been exploited to map the cross-over points between ts mutants of Ad5 and Ad2+ ND, (9, 14). In these studies, ts+ recombinants were isolated from cells coinfected with ts mutants of both Ad2+ ND1 and Ad5. In each case, the recombinant had Ad5 sequences at the site of the Ad2+ ND, ts lesion and Ad2+ ND, sequences at the site of the Ad5 ts lesion, thus explaining the ts+ phenotype. Similar mapping studies have been done with ts mutants of Ad2 and Ad5 (10).
In the present communication, we describe a simple method to construct recombinants between Ad2 and Ad5 with wild-type DNA restriction fragments with overlapping sequence homology. By using this method, we have constructed a number of recombinants between a large-plaque (Ip) mutant of Ad2 and wild-type Ad5 and localized the Ip mutation on the Ad2 genome. Our method for construction of recombinants between Ad2 and Ad5 will be valuable in studying aspects of adenovirus recombination as well as in experimnents involving genetic manipulations of the viral genome.
MATERIALS AND METHODS Cells and viruses. Human cell lines KB and 293 were grown as described previously (2). The 293 cells are a line of human embryo kidney cells transformed by transfection with sheared Ad5 DNA (5). Initial stocks of Ad2 and Ad5 were obtained from M. Green. The virus was propagated in KB cells grown in Spinner cultures. Virus was purified by the method of Green and Pina (7), and the DNA was extracted essentially as described elsewhere (8) with minor modifications. Transfection. DNA transfections were carried out on 293 cells by the "calcium-phosphate-precipitation" method (6) as described by Chinnadurai et al. (2). The 293 cells were used for transfection because the infectivity of Ad2 DNA is about 50- to 100-fold higher on 293 cells than on KB cells (2, 5). In many cases, the cell monolayers were treated with 20% Me2SO (2, 16). Isolation of plaque morphology mutant of Ad2. Ad2 virus was mutagenized with 1 M hydroxylamine in phosphate-buffered saline for 16 h. Mutagenized virus was dialyzed against phosphate-buffered saline
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and plaque assayed on KB cells at 33°C (2). Four wellseparated large plaques were isolated and replaqued. These isolates have been designated Ipl through Ip4. Isolation and screening of Ad2-Ad5 recombinants. Individual plaques from 293 dishes transfected with Ad2 and Ad5 DNA fragments were aspirated with Pasteur pipettes into 1.0 ml of growth medium containing 2% calf serum. Stocks of each plaque isolate were prepared by inoculating 0.2 ml of each plaque suspension into a 60-mm dish containing about 2 x 10' cells of 293 or KB cells. After complete cytopathic effect was observed, cells from each dish were collected along with the growth medium, sonicated, clarified by low-speed centrifugation, and stored at -70°C. To prepare 32P-labeled viral DNA, about 106 cells (KB or 293) in 35-mm dishes were infected with 0.05 to 0.1 ml of each virus stock. The infected cells were labeled with carrier-free H332PO4 (250 ,Ci/ml) from 20 to 36 h after infection. Viral DNA was selectively extracted by a modification of the method of Hirt (11). Infected cells were lyzed with 0.8 ml of a solution containing 0.6% sodium dodecyl sulfate, 10 mM Trishydrochloride (pH 7.4), and 10 mM EDTA and treated with 500 ,ug of pronase at 37°C for 1 h. High-molecularweight cellular DNA was precipitated with 0.2 ml of 5 M NaCl at 4°C overnight. Virus DNA was separated from cellular DNA by centrifugation at 15,000 x g for 20 min. The supernatant containing the virus DNA was extracted twice with phenol and dialyzed against 10 mM Tris-hydrochloride (pH 7.9) containing 1 mM EDTA. Large quantities of virus DNA was also prepared from virus purified by banding in two subsequent CsCl equilibrium density gradients (7). Digestion of viral DNA with restriction endonucleases and analysis by agarose gel electrophoresis. Virus DNA was digested with restriction endonuclease EcoRI (Miles Laboratories) in 100 mM Tris-hydrochloride (pH 7.9)-0.05 M KCl-10 mM MgCl2-0.1 mM EDTA. Digestion of virus DNA with Sall (New England Biolabs) was carried out in 150 mM NaCl, 7 mM Tris-hydrochloride (pH 7.4), 7 mM MgCl2, and 200 ,ug of bovine serum albumin per ml. Digestion with BamHI (Bethesda Research Laboratories) was carried out in 20 mM Tris-hydrochloride (pH 7.4), 7 mM MgCl2, and 7 mM ,8-mercaptoethanol. Digestion with HindIII (New England Biolabs) was carried out in 60 mM NaCl, 7 mM Tris-hydrochloride (pH 7.9), and 7 mM MgCl2. The enzyme reactions were terminated with 20 mM EDTA. Analytical slab gel electrophoresis was carried out in 1 or 1.4% agarose gels or in 2.2% acrylamide-0.7% agarose composite gels (1.5 by 20 by 40 mm). The gels were stained with 0.5 Mg of ethidium bromide per ml (15) and photographed. When 32P-labeled DNA was used, gels were air dried and autoradiographed with Kodak DF-85 X-ray film.
EcoRI may be due to the low levels of undigested DNA. Ad2 DNA is cleaved by EcoRI five times to yield six fragments (13; Fig. 1). Ad2 or Ad5 DNA is cleaved by Sall three times to yield four fragments (3, 4). We wished to determine whether cotransfection of cells with large overlapping DNA restriction fragments (EcoRI-A and Sall-A) would result in in vivo recombination between these fragments resulting in the production of infectious virus. As seen in Table 1, when Ad2 DNA was digested with EcoRI and cotransfected with Ad2 or Ad5 DNA digested with SalI, considerable infectivity could be seen (3 to 9 PFU/,ug of genome equivalent). It is noteworthy that cotransfection of Ad2 DNA digested with EcoRI and Sall gave consistently more plaques (5 to 9 PFU/j,g) compared with Ad5 DNA digested with SalI (3 to 6 PFU/,ug). To check whether the infectivity is due to true genetic recombination between the restriction fragments, 15 plaques were selected at random from dishes (in experiments 3 and 4) transfected with Ad2 and Ad5 DNA fragments and multiplied, and the DNA was analyzed by digestion with restriction endonucleases BamHI and EcoRI. Ad2 DNA is cleaved by BamHI at map positions 30, 42, and 59, making four fragments, whereas Ad5 DNA is cleaved only once at map position 59.0 (12; Fig. 1). Therefore, this enzyme is suitable for discriminating the Ad2 and Ad5 genome on the left half of the recombinant DNA. The right half of the recombinant DNA, on the other hand, could be easily identified as TABLE 1. Infectivity of restriction fragments of Ad2 and Ad5 DNAs on 293 cellsa Expt Enxopt 1
2
3
RESULTS
Infectivity of restriction fragments of Ad2 and Ad5 with overlapping sequence homology. Digestion of DNA digested with EcoRI or Sall virtually abolished transfection infectivity in 293 cells (Table 1). The low level of infectivity seen with DNA digested with
J. VIROL.
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of plaques per dish 137, 117 1,0
DNA
(,tg/dish)
Undigested Ad2 Ad2, EcoRI digested Ad2, SalI digested Ad2, EcoRI digested + Ad2, SalI digested
(0.4) (4.0) (4.0) (2.0 + 2.0)
Ad2, EcoRI digested Ad2, SalI digested Ad2, EcoRI digested + Ad2, SalI digested
(4.0) (4.0) (4.0 + 4.0)
0,1 0, 0 34, 20
Ad2, EcoRI digested Ad5, Sail digested Ad2, EcoRI digested + Ad5, SalI digested
(3.0) (2.5) (3.0 + 2.5)
0,0 0,0 12, 8
Ad2, EcoRI digested (2.5) Ad5, Sail digested (3.0) (2.5 + 3.0) Ad2, EcoRI digested + Ad5, SalI digested Transfection of the DNA was carried out by precipitation method (4) followed by an Me2SO described elsewhere (2). 4
0,0 12,13
0,0 0,0 15,12 the Ca2` boost as
LARGE-PLAQUE MUTANT OF Ad2
VFOL. 32, 1979 0
10
20
30
40
50
60
70
80
90
100
B F D E C A Ad2 !~~~~~~~~~~~~~~~~ *coR I E( Ad5 i B A C A C B D Ad2 ialI Ad5 C A D B Ad2 : B,3amHI Ad5 B A HL D G K F C B A I J E Ad2 Hiind m Ad5 F I G E C H D A B
FIG. 1. Maps of restriction endonuclease cleavage sites in Ad2 and Ad5 DNA. The cleavage maps for BamHI, EcoRI, and HindIII have been published by Sambrook et al. (14). The maps for SailI cleavage sites in Ad2 and Ad5 DNA have been published by Chow et al. (3) and Frost and Williams (4), respectively.
Ad2 or Ad5 by digestion with EcoRI. This enzyme cleaves Ad2 DNA five times and Ad5 DNA two times (12-14). All the EcoRI cleavage sites are located on the right half of the genome. Analysis of the DNA from 15 recombinants after cleavage with EcoRI and BamHI revealed that all the recombinants had identical structures, i.e., EcoRI generated three fragments similar to Ad5 parental DNA and BamHI generated four fragments similar to Ad2 parental DNA. The representative pattern of one of these recombinants is given in Fig. 2. These results indicate that the left half of the recombinant DNA is derived from Ad2 and the right half is derived from Ad5; recombination apparently occurred somewhere within position 42.0 and 58.5 between the Ad2 EcoRI-A fragment and Ad5 Sal-A fragment. Physical mapping of an Ip mutant of Ad2. We have isolated mutants of Ad2 by mutagenization with hydroxylamine which produce large clear plaques on KB cells in 7 to 8 days compared with the wild type which produces small diffused plaques in 10 to 12 days. The Ip mutants described in the present communication are similar in phenotype to the cyt mutants of Ad12 isolated by Takemori et al. (18). The Ip mutants of Ad2 exhibit the large-plaque phenotype at 33°C as well as at 37°C. However, the difference between the wild type and the mutant is greatly pronounced at 33°C (Fig. 3). Our DNA fragment transfection method for construction ofrecombinants is particularly suitable for construction of recombinants between the Ad2 Ip mutant and the Ad5 wild type and for mapping the site of the Ip lesion. This experiment is not practical with mixed infections of virions or intact DNAs because recombinants cannot be distinguished from the large excess of
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b BamH I
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B
A B
C
,.
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2 2 3 3 FIG. 2. Gel electrophoresis of Ad2, Ad5, and a recombinant DNA after cleavage with restriction endonucleases EcoRI and BamHI. Electrophoresis was carried out in 1% agarose gels as described elsewhere (2). Lane 1, Ad2; lane 2, Ad5; and lane 3, recombinant.
parental plaques that would be produced. Since the infectivity of the parental DNA is abolished by digestion with restriction endonucleases and only the recombinants can yield plaques, we have constructed several recombinants between an Ip mutant (Ip3) and the Ad5 wild type. These recombinants were used to physically map the Ip mutation by the approach described by Grodzicker et al. (9). From two experiments in which Ad2 Ip3 DNA digested with EcoRI and Ad5 wild-type DNA digested with SailI were cotransfected, 10 wellseparated plaques were isolated and multiplied and the DNA was analyzed by digestion with BamHI and EcoRI. All 10 isolates were found to be true recombinants and to exhibit the lp phenotype. Two types of recombinants were seen. Eight isolates (type I) had BamHI cleavage patterns identical to Ad2 and EcoRI cleavage patterns identical to Ad5 (Fig. 4). Two isolates (type II), after cleavage with BamHI, yielded one fragment similar in size to the Ad5 B or Ad2 A fragment and two other fragments similar in size to Ad2 B fragment (Fig. 4, right side, lane 4). Cleavage of DNA from type II recombinants with EcoRI yielded three fragments identical to Ad5 EcoRI fragments (Fig. 4a, lane 4). These results indicate that all the recombinants might
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FIG. 3. Plaque morphology of Ad2 wild type and Ad2 lp3 mutant. The wild type and the mutant viruses were plaque assayed on KB cells at 33°C (2) and stained with neutral red. The dishes were photographed with Polaroid film type 55 on day 15. (A) Ad2 wild type. (B) Ad2 lp3. -. .,
et
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45 FIG. 4. Autoradiogram of Ad2, Ad5, Ad2 lp3, and recombinant DNAs after cleavage with the restriction endonucleases BamHI and EcoRL DNA fragments were resolved in 1% agarose gels and autoradiographed as described in the text. Lane 1, Ad2 wild type; lane 2, Ad2 lp3; lane 3, type I recombinant of Ad5 and Ad2 lp3; lane 4, type II recombinant of Ad5 and Ad2 lp3; lane 5, Ad5 wild type.
have been generated by recombination between crossover for type I recombinants may be loEcoRI-A of Ad2 Ip3 DNA and SalI-A or SalI-A cated within map positions 42 to 59 (Fig. 1) since to -C (see below) of Ad5 DNA. The points of the DNA of these recombinants contain the Ad2
VOL. 32, 1979
LARGE-PLAQUE MUTANT OF Ad2
BamHI cleavage sites located at map positions 30 and 42. On the other hand, sites of crossover for type II recombinants may be located within map position 30 and 42 because the Ad2 BamHI cleavage site at map position 42 (Fig. 1) is absent in the recombinant DNA, but the cleavage site at map position 30 is present. The simplest mode by which these recombinants may have arisen is by a single recombination event between EcoRIA of Ad2 Ip3 DNA and either small portions of Ad5 DNA consisting of Sall-A to -C; these latter molecules may have been produced by incomplete digestion with Sall or may represent a subpopulation that has lost the SailI cleavage site at position 46. Cleavage of type II recombinant DNA with Sall yielded the characteristic Ad2 or Ad5 pattern, i.e., all four fragments were produced (data not shown), indicating that the former interpretation may be correct. All 10 recombinants selected were of Ip phenotype indicating that Ip mutation of Ad2 is located anywhere between map positions 0 to 42. To further narrow down the location of the Ip mutation, type II recombinant DNA was digested with restriction endonuclease HindIII (Fig. 5) and compared with Ad2 and Ad5 DNA fragments generated by HindIII. Within the left half of the genome, HindIII cleaves Ad2 but not Ad5 DNA at position 41 (Fig. 1). The recombinant genome had identical HindIII cleavage pattern as Ad5 wild type, i.e., Ad2 J fragment was not produced indicating that the sequences right of map position 41 are Ad5 and the sequences on the left are Ad2. Therefore, the Ip mutation is located within the left 41% of Ad2 genome. DISCUSSION We have described a novel method for the construction of recombinants between Ad2 and Ad5. This method involves cotransfection of DNA restriction fragments with overlapping sequences from the two parental DNAs to generate in vivo recombinant DNAs resulting in infectious virus. The restriction fragments with overlapping sequence homology have been shown to undergo specific in vivo recombination to produce infectious DNA. In principle, this method could be applied to other adenoviruses that undergo genetic recombination. This approach could also be used to construct recombinants having the desired segments of the two parental DNAs by transfection of appropriate restriction fragments. Such recombinants with defined segments of the two parental DNAs will be valuable in various genetic studies. We have successfully exploited this method to isolate adenovirus mutants lacking specific restriction sites (S. Rajagopalan and G. Chinnadurai, manuscript in preparation).
- A
A BC
627
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FIG. 5. Gel electrophoresis of Ad2, Ad5, and type II recombinant after cleavage with restriction endonuclease HindIII. Fragments were resolved by electrophoresis on 2.2% acrylamide gels containing 0.7% agarose (12) and photographed. Lane 1, Ad2 wild type; lane 2, type II recombinant; lane 3, Ad5 wild type.
Our results indicate that genetic recombination may occur in the absence of viral DNA replication. It is likely that the recombination is carried out by the host enzymes or perhaps by virus proteins coded by the discontinuous virus genome. This method could be used to investigate aspects of adenovirus and cellular DNA recombination. We have constructed several recombinants between an Ip mutant of Ad2 and Ad5 wild type. By dissection of the recombinant genomes with restriction endonucleases that cleave Ad2 and Ad5 differently, we have localized the Ip mutation to the left 41% of Ad2 genome. It is not possible at present to more precisely map the Ip lesion within the left 41% of the viral genome, because no restriction endonucleases have been described that cleave Ad2 and Ad5 differently within this region. The mapping approach that we have used was developed by Sambrook, Williams, and co-workers (9, 14) for physical mapping of the ts mutants of Ad2+NDj and Ad5 and has recently been used for physical mapping of ts mutants of Ad2 (10). Recently, ts mutants of Ad5 have been mapped by a marker rescue technique by co-
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CHINNADURAI, CHINNADURAI, AND BRUSCA
transfection of wild-type DNA fragments and ts mutant DNA (1, 4). Application of modified versions of this approach for physical mapping of plaque morphology mutants of Ad2 has yet to be exploited. In the left 41% of Ad2 and Ad5 genome several ts and two host range (hr) mutants have been mapped (1, 4, 10). One of the ts mutants, H5ts36, and two hr mutants have lesions in early genes, whereas the other ts mutants, H5ts49, H5ts58, and H2ts4, are defective in late genes. It will be of interest to see whether the Ip mutation is in an early or late gene. ACKNOWLEDGMENTS This investigation was supported by research grants from the National Science Foundation (PCM77-12662), Missouri Cancer Programs, Inc., and an institutional grant from the American Cancer Society (77005). G.C. is an Established Investigator of the American Heart Association. We thank Maurice Green for support and encouragement, G. Gerard and W. S. M. Wold for critical reading of the manuscript, and Eric Frost for supplying the Ad5 SalI cleavage map before publication. LITERATURE CITED 1. Arrand, J. E. 1978. Mapping of adenovirus type 5 temperature-sensitive mutations by marker rescue in enhanced double DNA infections. J. Gen. Virol. 41:573586. 2. Chinnadurai, G., S. Chinnadurai, and M. Green. 1978. Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex. J. Virol. 26:195-199. 3. Chow, L. R., J. M. Roberts, J. B. Lewis, and T. R. Booker. 1977. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA-DNA hybrids. Cell 11:819-836. 4. Frost, E., and J. Williams. 1978. Mapping of temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology 91:39-50. 5. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977. Characteristics of human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36: 59-74. 6. Graham, F. L., and A. J. van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456-467.
J. VIROL.
7. Green, M., and M. Pina. 1963. Biochemical studies on /adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20:199207. 8. Green, M., and M. Pina. 1964. Biochemical studies on adenovirus multiplication. VI. Properties of highly purified tumorigenic human adenoviruses and their DNAs. Proc. Natl. Acad. Sci. U.S.A. 51:1251-1259. 9. Grodzicker, T., J. Williams, P. Sharp, and J. Sambrook. 1974. Physical mapping of temperature-sensitive mutations of adenoviruses. Cold Spring Harbor Symp. Quant. Biol. 39:439-446. 10. Hassell, J. A., and J. Weber. 1978. Genetic analysis of adenovirus type 2. VIII. Physical locations of temperature-sensitive mutations. J. Virol. 28:671-678. 11. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. 12. Mulder, C., J. R. Arrand, H. Delius, W. Keller, U. Pettersson, R. J. Roberts, and P. A. Sharp. 1974. Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases EcoRI and HpaI. Cold Spring Harbor Symp. Quant. Biol. 39:397-400. 13. Pettersson, U., C. Mulder, H. Delius, and P. A. Sharp. 1973. Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R.RI. Proc. Natl. Acad. Sci. U.S.A. 70:200-204. 14. Sambrook, J., J. Williams, P. A. Sharp, and T. Grodzicker. 1975. Physical mapping of temperature sensitive mutations of adenoviruses. J. Mol. Biol. 97:369390. 15. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose ethidium bromide electrophoresis. Biochemistry 12: 3055-3063. 16. Stow, N. D., and N. M. Wilkie. 1976. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J. Gen. Virol. 33: 447-458. 17. Takemori, N. 1972. Genetic studies with tumorigenic adenoviruses. III. Recombination in adenovirus type 12. Virology 47:157-167. 18. Takemori, N., J. L. Riggs, and C. H. Aldrich. 1968. Genetic studies with tumorigenic adenoviruses. I. Isolation of cytocidal (cyt) mutants of adenovirus type 12. Virology 36:575-586. 19. Williams, J., C. Young, and P. Austin. 1974. Genetic analysis of human adenovirus type 5 in permissive and non-permissive cells. Cold Spring Harbor Symp Quant. Biol. 39:427-437.
OF
Vol. 32, No. 2
VIROLOGY, Nov. 1979, p. 623-628
0022-538X/79/11-0623/06$02.00/0
Physical Mapping of a Large-Plaque Mutation of Adenovirus Type 2 G. CHINNADURAI,* SHANBAGAM CHINNADURAI, AND JOHN BRUSCA Institute for Molecular Virology, Saint Louis University School of Medicine, St. Louis, Missouri 63110 Received for publication 2 April 1979
We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 Sail-A fragment. By using this method, several recombinants were constructed between a large-plaque (Ip) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the Ip mutation is located within the left 41% of Ad2 genome.
The 31 recognized human adenovirus serotypes form five groups (A to E) based upon genomic DNA homologies in molecular hybridization experiments (M. Green, J. K. Mackey, W. S. M. Wold, and P. Rigden, Virology, in press). The DNA homologies of serotypes within each group range from 48 to 69% (group A) to 99 to 100% (group C). Because of this group-specific DNA homology, recombination between serotypes might be expected to occur during infections. Takemori (17) first demonstrated genetic recombination between cytocidal (cyt) mutants of adenovirus type 12 (Adl2) (group A). Subsequently, Williams and co-workers (19) demonstrated recombination among temperature-sensitive (ts) mutants of Ad5 (group C) and constructed a genetic map of the Ad5 genome based upon recombination frequencies. Although the genomes of Ad2 and Ad5 are similar, minor differences in base sequences exist because some restriction endonucleases generate different fragments from Ad2 and Ad5 DNA. These differences in cleavage patterns have been exploited to map the cross-over points between ts mutants of Ad5 and Ad2+ ND, (9, 14). In these studies, ts+ recombinants were isolated from cells coinfected with ts mutants of both Ad2+ ND1 and Ad5. In each case, the recombinant had Ad5 sequences at the site of the Ad2+ ND, ts lesion and Ad2+ ND, sequences at the site of the Ad5 ts lesion, thus explaining the ts+ phenotype. Similar mapping studies have been done with ts mutants of Ad2 and Ad5 (10).
In the present communication, we describe a simple method to construct recombinants between Ad2 and Ad5 with wild-type DNA restriction fragments with overlapping sequence homology. By using this method, we have constructed a number of recombinants between a large-plaque (Ip) mutant of Ad2 and wild-type Ad5 and localized the Ip mutation on the Ad2 genome. Our method for construction of recombinants between Ad2 and Ad5 will be valuable in studying aspects of adenovirus recombination as well as in experimnents involving genetic manipulations of the viral genome.
MATERIALS AND METHODS Cells and viruses. Human cell lines KB and 293 were grown as described previously (2). The 293 cells are a line of human embryo kidney cells transformed by transfection with sheared Ad5 DNA (5). Initial stocks of Ad2 and Ad5 were obtained from M. Green. The virus was propagated in KB cells grown in Spinner cultures. Virus was purified by the method of Green and Pina (7), and the DNA was extracted essentially as described elsewhere (8) with minor modifications. Transfection. DNA transfections were carried out on 293 cells by the "calcium-phosphate-precipitation" method (6) as described by Chinnadurai et al. (2). The 293 cells were used for transfection because the infectivity of Ad2 DNA is about 50- to 100-fold higher on 293 cells than on KB cells (2, 5). In many cases, the cell monolayers were treated with 20% Me2SO (2, 16). Isolation of plaque morphology mutant of Ad2. Ad2 virus was mutagenized with 1 M hydroxylamine in phosphate-buffered saline for 16 h. Mutagenized virus was dialyzed against phosphate-buffered saline
623
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CHINNADURAI, CHINNADURAI, AND BRUSCA
and plaque assayed on KB cells at 33°C (2). Four wellseparated large plaques were isolated and replaqued. These isolates have been designated Ipl through Ip4. Isolation and screening of Ad2-Ad5 recombinants. Individual plaques from 293 dishes transfected with Ad2 and Ad5 DNA fragments were aspirated with Pasteur pipettes into 1.0 ml of growth medium containing 2% calf serum. Stocks of each plaque isolate were prepared by inoculating 0.2 ml of each plaque suspension into a 60-mm dish containing about 2 x 10' cells of 293 or KB cells. After complete cytopathic effect was observed, cells from each dish were collected along with the growth medium, sonicated, clarified by low-speed centrifugation, and stored at -70°C. To prepare 32P-labeled viral DNA, about 106 cells (KB or 293) in 35-mm dishes were infected with 0.05 to 0.1 ml of each virus stock. The infected cells were labeled with carrier-free H332PO4 (250 ,Ci/ml) from 20 to 36 h after infection. Viral DNA was selectively extracted by a modification of the method of Hirt (11). Infected cells were lyzed with 0.8 ml of a solution containing 0.6% sodium dodecyl sulfate, 10 mM Trishydrochloride (pH 7.4), and 10 mM EDTA and treated with 500 ,ug of pronase at 37°C for 1 h. High-molecularweight cellular DNA was precipitated with 0.2 ml of 5 M NaCl at 4°C overnight. Virus DNA was separated from cellular DNA by centrifugation at 15,000 x g for 20 min. The supernatant containing the virus DNA was extracted twice with phenol and dialyzed against 10 mM Tris-hydrochloride (pH 7.9) containing 1 mM EDTA. Large quantities of virus DNA was also prepared from virus purified by banding in two subsequent CsCl equilibrium density gradients (7). Digestion of viral DNA with restriction endonucleases and analysis by agarose gel electrophoresis. Virus DNA was digested with restriction endonuclease EcoRI (Miles Laboratories) in 100 mM Tris-hydrochloride (pH 7.9)-0.05 M KCl-10 mM MgCl2-0.1 mM EDTA. Digestion of virus DNA with Sall (New England Biolabs) was carried out in 150 mM NaCl, 7 mM Tris-hydrochloride (pH 7.4), 7 mM MgCl2, and 200 ,ug of bovine serum albumin per ml. Digestion with BamHI (Bethesda Research Laboratories) was carried out in 20 mM Tris-hydrochloride (pH 7.4), 7 mM MgCl2, and 7 mM ,8-mercaptoethanol. Digestion with HindIII (New England Biolabs) was carried out in 60 mM NaCl, 7 mM Tris-hydrochloride (pH 7.9), and 7 mM MgCl2. The enzyme reactions were terminated with 20 mM EDTA. Analytical slab gel electrophoresis was carried out in 1 or 1.4% agarose gels or in 2.2% acrylamide-0.7% agarose composite gels (1.5 by 20 by 40 mm). The gels were stained with 0.5 Mg of ethidium bromide per ml (15) and photographed. When 32P-labeled DNA was used, gels were air dried and autoradiographed with Kodak DF-85 X-ray film.
EcoRI may be due to the low levels of undigested DNA. Ad2 DNA is cleaved by EcoRI five times to yield six fragments (13; Fig. 1). Ad2 or Ad5 DNA is cleaved by Sall three times to yield four fragments (3, 4). We wished to determine whether cotransfection of cells with large overlapping DNA restriction fragments (EcoRI-A and Sall-A) would result in in vivo recombination between these fragments resulting in the production of infectious virus. As seen in Table 1, when Ad2 DNA was digested with EcoRI and cotransfected with Ad2 or Ad5 DNA digested with SalI, considerable infectivity could be seen (3 to 9 PFU/,ug of genome equivalent). It is noteworthy that cotransfection of Ad2 DNA digested with EcoRI and Sall gave consistently more plaques (5 to 9 PFU/j,g) compared with Ad5 DNA digested with SalI (3 to 6 PFU/,ug). To check whether the infectivity is due to true genetic recombination between the restriction fragments, 15 plaques were selected at random from dishes (in experiments 3 and 4) transfected with Ad2 and Ad5 DNA fragments and multiplied, and the DNA was analyzed by digestion with restriction endonucleases BamHI and EcoRI. Ad2 DNA is cleaved by BamHI at map positions 30, 42, and 59, making four fragments, whereas Ad5 DNA is cleaved only once at map position 59.0 (12; Fig. 1). Therefore, this enzyme is suitable for discriminating the Ad2 and Ad5 genome on the left half of the recombinant DNA. The right half of the recombinant DNA, on the other hand, could be easily identified as TABLE 1. Infectivity of restriction fragments of Ad2 and Ad5 DNAs on 293 cellsa Expt Enxopt 1
2
3
RESULTS
Infectivity of restriction fragments of Ad2 and Ad5 with overlapping sequence homology. Digestion of DNA digested with EcoRI or Sall virtually abolished transfection infectivity in 293 cells (Table 1). The low level of infectivity seen with DNA digested with
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of plaques per dish 137, 117 1,0
DNA
(,tg/dish)
Undigested Ad2 Ad2, EcoRI digested Ad2, SalI digested Ad2, EcoRI digested + Ad2, SalI digested
(0.4) (4.0) (4.0) (2.0 + 2.0)
Ad2, EcoRI digested Ad2, SalI digested Ad2, EcoRI digested + Ad2, SalI digested
(4.0) (4.0) (4.0 + 4.0)
0,1 0, 0 34, 20
Ad2, EcoRI digested Ad5, Sail digested Ad2, EcoRI digested + Ad5, SalI digested
(3.0) (2.5) (3.0 + 2.5)
0,0 0,0 12, 8
Ad2, EcoRI digested (2.5) Ad5, Sail digested (3.0) (2.5 + 3.0) Ad2, EcoRI digested + Ad5, SalI digested Transfection of the DNA was carried out by precipitation method (4) followed by an Me2SO described elsewhere (2). 4
0,0 12,13
0,0 0,0 15,12 the Ca2` boost as
LARGE-PLAQUE MUTANT OF Ad2
VFOL. 32, 1979 0
10
20
30
40
50
60
70
80
90
100
B F D E C A Ad2 !~~~~~~~~~~~~~~~~ *coR I E( Ad5 i B A C A C B D Ad2 ialI Ad5 C A D B Ad2 : B,3amHI Ad5 B A HL D G K F C B A I J E Ad2 Hiind m Ad5 F I G E C H D A B
FIG. 1. Maps of restriction endonuclease cleavage sites in Ad2 and Ad5 DNA. The cleavage maps for BamHI, EcoRI, and HindIII have been published by Sambrook et al. (14). The maps for SailI cleavage sites in Ad2 and Ad5 DNA have been published by Chow et al. (3) and Frost and Williams (4), respectively.
Ad2 or Ad5 by digestion with EcoRI. This enzyme cleaves Ad2 DNA five times and Ad5 DNA two times (12-14). All the EcoRI cleavage sites are located on the right half of the genome. Analysis of the DNA from 15 recombinants after cleavage with EcoRI and BamHI revealed that all the recombinants had identical structures, i.e., EcoRI generated three fragments similar to Ad5 parental DNA and BamHI generated four fragments similar to Ad2 parental DNA. The representative pattern of one of these recombinants is given in Fig. 2. These results indicate that the left half of the recombinant DNA is derived from Ad2 and the right half is derived from Ad5; recombination apparently occurred somewhere within position 42.0 and 58.5 between the Ad2 EcoRI-A fragment and Ad5 Sal-A fragment. Physical mapping of an Ip mutant of Ad2. We have isolated mutants of Ad2 by mutagenization with hydroxylamine which produce large clear plaques on KB cells in 7 to 8 days compared with the wild type which produces small diffused plaques in 10 to 12 days. The Ip mutants described in the present communication are similar in phenotype to the cyt mutants of Ad12 isolated by Takemori et al. (18). The Ip mutants of Ad2 exhibit the large-plaque phenotype at 33°C as well as at 37°C. However, the difference between the wild type and the mutant is greatly pronounced at 33°C (Fig. 3). Our DNA fragment transfection method for construction ofrecombinants is particularly suitable for construction of recombinants between the Ad2 Ip mutant and the Ad5 wild type and for mapping the site of the Ip lesion. This experiment is not practical with mixed infections of virions or intact DNAs because recombinants cannot be distinguished from the large excess of
a EcoR I
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b BamH I
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A B
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,.
D:;.
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2 2 3 3 FIG. 2. Gel electrophoresis of Ad2, Ad5, and a recombinant DNA after cleavage with restriction endonucleases EcoRI and BamHI. Electrophoresis was carried out in 1% agarose gels as described elsewhere (2). Lane 1, Ad2; lane 2, Ad5; and lane 3, recombinant.
parental plaques that would be produced. Since the infectivity of the parental DNA is abolished by digestion with restriction endonucleases and only the recombinants can yield plaques, we have constructed several recombinants between an Ip mutant (Ip3) and the Ad5 wild type. These recombinants were used to physically map the Ip mutation by the approach described by Grodzicker et al. (9). From two experiments in which Ad2 Ip3 DNA digested with EcoRI and Ad5 wild-type DNA digested with SailI were cotransfected, 10 wellseparated plaques were isolated and multiplied and the DNA was analyzed by digestion with BamHI and EcoRI. All 10 isolates were found to be true recombinants and to exhibit the lp phenotype. Two types of recombinants were seen. Eight isolates (type I) had BamHI cleavage patterns identical to Ad2 and EcoRI cleavage patterns identical to Ad5 (Fig. 4). Two isolates (type II), after cleavage with BamHI, yielded one fragment similar in size to the Ad5 B or Ad2 A fragment and two other fragments similar in size to Ad2 B fragment (Fig. 4, right side, lane 4). Cleavage of DNA from type II recombinants with EcoRI yielded three fragments identical to Ad5 EcoRI fragments (Fig. 4a, lane 4). These results indicate that all the recombinants might
626
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CHINNADURAI, CHINNADURAI, AND BRUSCA
FIG. 3. Plaque morphology of Ad2 wild type and Ad2 lp3 mutant. The wild type and the mutant viruses were plaque assayed on KB cells at 33°C (2) and stained with neutral red. The dishes were photographed with Polaroid film type 55 on day 15. (A) Ad2 wild type. (B) Ad2 lp3. -. .,
et
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have been generated by recombination between crossover for type I recombinants may be loEcoRI-A of Ad2 Ip3 DNA and SalI-A or SalI-A cated within map positions 42 to 59 (Fig. 1) since to -C (see below) of Ad5 DNA. The points of the DNA of these recombinants contain the Ad2
VOL. 32, 1979
LARGE-PLAQUE MUTANT OF Ad2
BamHI cleavage sites located at map positions 30 and 42. On the other hand, sites of crossover for type II recombinants may be located within map position 30 and 42 because the Ad2 BamHI cleavage site at map position 42 (Fig. 1) is absent in the recombinant DNA, but the cleavage site at map position 30 is present. The simplest mode by which these recombinants may have arisen is by a single recombination event between EcoRIA of Ad2 Ip3 DNA and either small portions of Ad5 DNA consisting of Sall-A to -C; these latter molecules may have been produced by incomplete digestion with Sall or may represent a subpopulation that has lost the SailI cleavage site at position 46. Cleavage of type II recombinant DNA with Sall yielded the characteristic Ad2 or Ad5 pattern, i.e., all four fragments were produced (data not shown), indicating that the former interpretation may be correct. All 10 recombinants selected were of Ip phenotype indicating that Ip mutation of Ad2 is located anywhere between map positions 0 to 42. To further narrow down the location of the Ip mutation, type II recombinant DNA was digested with restriction endonuclease HindIII (Fig. 5) and compared with Ad2 and Ad5 DNA fragments generated by HindIII. Within the left half of the genome, HindIII cleaves Ad2 but not Ad5 DNA at position 41 (Fig. 1). The recombinant genome had identical HindIII cleavage pattern as Ad5 wild type, i.e., Ad2 J fragment was not produced indicating that the sequences right of map position 41 are Ad5 and the sequences on the left are Ad2. Therefore, the Ip mutation is located within the left 41% of Ad2 genome. DISCUSSION We have described a novel method for the construction of recombinants between Ad2 and Ad5. This method involves cotransfection of DNA restriction fragments with overlapping sequences from the two parental DNAs to generate in vivo recombinant DNAs resulting in infectious virus. The restriction fragments with overlapping sequence homology have been shown to undergo specific in vivo recombination to produce infectious DNA. In principle, this method could be applied to other adenoviruses that undergo genetic recombination. This approach could also be used to construct recombinants having the desired segments of the two parental DNAs by transfection of appropriate restriction fragments. Such recombinants with defined segments of the two parental DNAs will be valuable in various genetic studies. We have successfully exploited this method to isolate adenovirus mutants lacking specific restriction sites (S. Rajagopalan and G. Chinnadurai, manuscript in preparation).
- A
A BC
627
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FIG. 5. Gel electrophoresis of Ad2, Ad5, and type II recombinant after cleavage with restriction endonuclease HindIII. Fragments were resolved by electrophoresis on 2.2% acrylamide gels containing 0.7% agarose (12) and photographed. Lane 1, Ad2 wild type; lane 2, type II recombinant; lane 3, Ad5 wild type.
Our results indicate that genetic recombination may occur in the absence of viral DNA replication. It is likely that the recombination is carried out by the host enzymes or perhaps by virus proteins coded by the discontinuous virus genome. This method could be used to investigate aspects of adenovirus and cellular DNA recombination. We have constructed several recombinants between an Ip mutant of Ad2 and Ad5 wild type. By dissection of the recombinant genomes with restriction endonucleases that cleave Ad2 and Ad5 differently, we have localized the Ip mutation to the left 41% of Ad2 genome. It is not possible at present to more precisely map the Ip lesion within the left 41% of the viral genome, because no restriction endonucleases have been described that cleave Ad2 and Ad5 differently within this region. The mapping approach that we have used was developed by Sambrook, Williams, and co-workers (9, 14) for physical mapping of the ts mutants of Ad2+NDj and Ad5 and has recently been used for physical mapping of ts mutants of Ad2 (10). Recently, ts mutants of Ad5 have been mapped by a marker rescue technique by co-
628
CHINNADURAI, CHINNADURAI, AND BRUSCA
transfection of wild-type DNA fragments and ts mutant DNA (1, 4). Application of modified versions of this approach for physical mapping of plaque morphology mutants of Ad2 has yet to be exploited. In the left 41% of Ad2 and Ad5 genome several ts and two host range (hr) mutants have been mapped (1, 4, 10). One of the ts mutants, H5ts36, and two hr mutants have lesions in early genes, whereas the other ts mutants, H5ts49, H5ts58, and H2ts4, are defective in late genes. It will be of interest to see whether the Ip mutation is in an early or late gene. ACKNOWLEDGMENTS This investigation was supported by research grants from the National Science Foundation (PCM77-12662), Missouri Cancer Programs, Inc., and an institutional grant from the American Cancer Society (77005). G.C. is an Established Investigator of the American Heart Association. We thank Maurice Green for support and encouragement, G. Gerard and W. S. M. Wold for critical reading of the manuscript, and Eric Frost for supplying the Ad5 SalI cleavage map before publication. LITERATURE CITED 1. Arrand, J. E. 1978. Mapping of adenovirus type 5 temperature-sensitive mutations by marker rescue in enhanced double DNA infections. J. Gen. Virol. 41:573586. 2. Chinnadurai, G., S. Chinnadurai, and M. Green. 1978. Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex. J. Virol. 26:195-199. 3. Chow, L. R., J. M. Roberts, J. B. Lewis, and T. R. Booker. 1977. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA-DNA hybrids. Cell 11:819-836. 4. Frost, E., and J. Williams. 1978. Mapping of temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology 91:39-50. 5. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977. Characteristics of human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36: 59-74. 6. Graham, F. L., and A. J. van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456-467.
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7. Green, M., and M. Pina. 1963. Biochemical studies on /adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20:199207. 8. Green, M., and M. Pina. 1964. Biochemical studies on adenovirus multiplication. VI. Properties of highly purified tumorigenic human adenoviruses and their DNAs. Proc. Natl. Acad. Sci. U.S.A. 51:1251-1259. 9. Grodzicker, T., J. Williams, P. Sharp, and J. Sambrook. 1974. Physical mapping of temperature-sensitive mutations of adenoviruses. Cold Spring Harbor Symp. Quant. Biol. 39:439-446. 10. Hassell, J. A., and J. Weber. 1978. Genetic analysis of adenovirus type 2. VIII. Physical locations of temperature-sensitive mutations. J. Virol. 28:671-678. 11. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. 12. Mulder, C., J. R. Arrand, H. Delius, W. Keller, U. Pettersson, R. J. Roberts, and P. A. Sharp. 1974. Cleavage maps of DNA from adenovirus types 2 and 5 by restriction endonucleases EcoRI and HpaI. Cold Spring Harbor Symp. Quant. Biol. 39:397-400. 13. Pettersson, U., C. Mulder, H. Delius, and P. A. Sharp. 1973. Cleavage of adenovirus type 2 DNA into six unique fragments by endonuclease R.RI. Proc. Natl. Acad. Sci. U.S.A. 70:200-204. 14. Sambrook, J., J. Williams, P. A. Sharp, and T. Grodzicker. 1975. Physical mapping of temperature sensitive mutations of adenoviruses. J. Mol. Biol. 97:369390. 15. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose ethidium bromide electrophoresis. Biochemistry 12: 3055-3063. 16. Stow, N. D., and N. M. Wilkie. 1976. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J. Gen. Virol. 33: 447-458. 17. Takemori, N. 1972. Genetic studies with tumorigenic adenoviruses. III. Recombination in adenovirus type 12. Virology 47:157-167. 18. Takemori, N., J. L. Riggs, and C. H. Aldrich. 1968. Genetic studies with tumorigenic adenoviruses. I. Isolation of cytocidal (cyt) mutants of adenovirus type 12. Virology 36:575-586. 19. Williams, J., C. Young, and P. Austin. 1974. Genetic analysis of human adenovirus type 5 in permissive and non-permissive cells. Cold Spring Harbor Symp Quant. Biol. 39:427-437.
