Proc. Nati. Acad. Sci. USA Vol. 89, pp. 9977-9981, November 1992 Biochemistry Construction of chimeric vaccinia by molecular cloning and packaging (recombinant DNA/orthopoxvfrus/avipoxvlrus/host-range selectlon/Gpt selection) FRIEDRICH SCHEIFLINGER, FRIEDRICH DORNER*, AND FALKO G. FALKNER IMMUNO A.G., Biomedical Research Center, Uferstrasse 15, A-2304 Orth/Donau, Austria Communicated by Max L. Birnstiel, July 29, 1992 (receivedfor review February 20, 1992)

ABSTRACT Foreign DNA was inserted into unique re- thopoxvirus DNA by viable orthopox helper (homolo- striction endonuclease cleavage sites (Sma I or Not I) of the gous packaging) was demonstrated (10). 200,000-base-pair vaccinia virus by direct molecular Heterologous packaging of genomic viral DNA-e.g., cloning. The modified vaccinia virus DNA was packed in packaging oforthopoxvirus DNA by viable avipoxvirus-has fowlpox virus-infected avian cells, and chimeric vaccinia virus not been demonstrated so far. We have developed methods was isolated from mammalian cells not supporting the growth by which in vitro modified genomic DNA can be packaged of the fowlpox helper virus. In contrast to the classical "in using an avipox (fowlpox) helper virus, in either primary vivo" recombination technique, chimeric viruses with inserts in chicken embryo fibroblasts (CEFs) or a continuous mamma- both possible orientations and families of chimeras with mul- lian line. The latter procedure circumvents the tedious tiple inserts were obtained. The different genomic configura- requirement for preparing primary chicken cell cultures for tions of chimeric viruses provide a broader basis for screening each packaging experiment and eliminates the helper virus of optimal viruses. In addition to p aging in avian cells, a from the initial virus stock. Packaged chimeric vaccinia virus second packaging procedure for vaccinia DNA, based on the was selected for by plaquing on mammalian cells. These cells abortive infection of mammalian cells with the fowlpox helper do not permit the growth ofthe helper virus, as fowlpox virus virus, was developed. This procedure permits simultaneous grows in avian cells only, whereas vaccinia virus (orthopox) packaging has a broad host range, growing in many vertebrate cells, and host-range selection for the packaged virus. The including avian cells. We report here the structures and the cloning/packaging procedure allows the direct insertion of properties ofthe different chimeric vaccinia viruses obtained foreign DNA without the need for having flanking by using this system. regions homologous to viral nonessential regions and is inde- pendent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% ofthe total vaccinia virus MATERIALS AND METHODS yield consisted of chimeras. The procedure is, therefore, a Cells and Viruses. Vaccinia virus (WR strain; ATCC VR useful tool in molecular . 119) and the African green monkey kidney cell line CV-1 (ATCC CCL 70) were obtained from the American Type Vaccinia virus is one of the largest viruses, with a Culture Collection. The attenuated fowlpox virus strain genome consisting of a single linear double-stranded DNA HP1.441 (11) was kindly provided by A. Mayr (Institut fur molecule of about 200,000 base pairs. Replication and tran- Medical Mikrobiologie, Tierarztliche Fakultit, Munich). scription occur in the cytoplasm of the host cell (1, 2). Vaccinia and fowlpox virus were purified by two successive Recombinant poxviruses are therefore constructed by "in sucrose gradients (12). CEFs were prepared from 12-day vivo" recombination techniques involving transfection of chicken embryos. The cells were grown in tissue culture appropriate plasmids into virus-infected cells (3, 4). With the medium 199 (GIBCO/BRL) supplemented with 5% fetal classical method, however, usually only one copy of the bovine serum, glutamine, and antibiotics. foreign is inserted into the viral genome in one defined Cloning of Genomic Vaccinia DNA. Cloning into the Sma I orientation. Since position and gene dosage effects influence site. Viral DNA was prepared according to ref. 13; care was transcription levels (5), a method that would lead to the taken to avoid shear forces. Vaccinia DNA was cleaved with integration of the foreign in different genomic config- Sma I and purified by one phenol and three chloroform urations is desirable. Chimeric viruses were therefore con- extractions. The integrity of the vaccinia vector arms was structed by direct molecular cloning and, because naked viral controlled by field-inversion gel electrophoresis (14). Two DNA is noninfectious when transfected into permissive cells, micrograms ofthe cleaved virus DNA was ligated with 400 ng by in vivo of the insert [the 1.1-kilobase (kb) Hpa I-Dra I fragment packaging. containing the gpt gene cassette excised from pTKgpt-Fls; Previous studies showed that it was possible to reactivate ref. 15] in 30 Al for 40 hr with 15 units of T4 DNA ligase denatured poxviruses by homologous or heterologous pox (resulting in the F12-series plaques). The second ligation helper viruses (6-9). This phenomenon, reported decades experiment (F13 series) was done under the same conditions, ago, was observed following coinfections with heat- except that a 17-fold molar excess of the 1.1-kb insert and 5 inactivated myxoma virus and viable fibroma virus (6). units of ligase were used. Treatment of viral suspensions with agents destroying the Cloning into the Not I site. Vaccinia DNA (1 ug) was viral core structure was found to prevent reactivation-i.e., cleaved with Not I and further treated as described above. the reactivation phenomenon was dependent on intact viral The insert, the 1.1-kb P7.5 gpt gene cassette flanked by Not substructures and could not be achieved with naked viral I sites, was prepared from the pN2-gpta (see below). DNA. In later studies, however, in vivo packaging of or- Ligation conditions were the same as described for the

The publication costs of this article were defrayed in part by page charge Abbreviations: CEF, chicken embryo fibroblast; pfu, plaque- payment. This article must therefore be hereby marked "advertisement" forming unit(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9977 Downloaded by guest on September 26, 2021 9978 Biochemistry: Scheiflinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) F12-series plaques. Various amounts ofinsert were used (see vaccinia protocols (17). The plaque assays and the gpt Table 1). selection were performed with CV-1 cells (15). Construction of the Plasmids. The plasmid pBluescript II Heterologous in Vivo Packaging in Mammalin Cells. Con- SK(-) (Stratagene) was digested with HincII and ligated with fluent monolayers of the African green monkey kidney line Not I linkers (Pharmacia). The resulting plasmid, pN2, has a CV-1 (6 x 106 cells) were abortively infected at 0.5 pfu per multiple cloning site (Not I, Xba I, Spe I, BamHI, Sma I, Pst cell with the fowlpox virus HP1.441. Transfection of the I, EcoRP, EcoRV, HindIII, Cla I, and Not I) flanked by Not ligated DNA, preparation of the crude viral stocks, and I sites. The 1.1-kb Hpa I-Dra I fragment (containing the gpt plaque assays were performed as described above. gene cassette; see above) was inserted into the Sma I site of pN2. The two resulting plasmids, with inserts in opposite RESULTS orientations, were designated pN2-gpta and pN2-gptb. Field-Inversion Gel Electrophoresis. The Sma I- or Not Insertion ofthe Foreign DNA and Heterologous Packaging in I-digested viral DNA was electrophoresed in a 1% agarose gel Avian Cells. To construct chimeric vaccinia viruses, we in 20 mM Tris/10 mM glacial acetic acid/0.5 mM EDTA, pH directly inserted a model gene cassette, the Escherichia coli 8.0 with a microcomputer-controlled power supply (Consort xanthine (guanine) phosphoribosyltransferase (gpt) gene model E790) in the cold room. To separate the whole range driven by the vaccinia virus P7.5 promoter (18), into the of four programs were run successively; in all single genomic Sma I site of the WR strain of vaccinia virus, fragments, as outlined in Fig. 1 and described in Materials and Methods. programs, the voltage was set at 7 V/cm; the forward (F) and The Sma I site is located in the nonessential open reading the reverse (R) pulse were followed by a pause (P) of 1 sec. frame A51R (19). Viral DNA was packaged in fowlpox The programs were as follows: 1, 4 hr F6 R3; 2, 4 hr F4 R2; virus-infected CEFs and chimeric vaccinia viruses were 3,4hrF2Rl;4,8hrF8R4. selected by their Gpt+ phenotypes in CV-1 cells. In the Heterologous in Vivo Packaging in Avian Ceils. Confluent F12-series experiment (see Materials and Methods) the titer monolayers of CEFs (6 x 106 cells) were infected with the ofpackaged chimeric virus in the crude stock was 9 x 102 pfu fowlpox virus HP1.441 at 0.5 plaque-forming unit (pfu) per per 6 x 106 cells, and in the F13-series experiment the titer cell and incubated for 1 hr. Ligated DNA (2 pzg) was trans- was 5 x 102 pfu per 6 x 106 cells. The fowlpox helper virus fected into the infected cells by the calcium phosphate usually produced titers of 108 pfu per 6 x 106 CEFs. In technique (16). After 3 days of incubation, the cells were contrast to the previously described genome rescue (homol- harvested and a crude viral stock was prepared by standard ogous packaging) of intact orthopoxvirus DNA by a related orthopox virus (10), in our packaging system the viral DNA SmaI to be packaged and the helper virus belong to different genera of the poxvirus family (to the orthopox and the avipoxvi- Vaccinia virus (WR) cleave ruses, respectively). This heterologous packaging procedure, genomic DNA (200 kb) combined with chemical selection, allowed the rapid isolation with SmaI of the packaged chimeric virus.

I'T, - r." -,,. -Z V-, r-i rl), r", r.-', r1l", C-', s o> E E z E E Ez = ligate rinsert .Ad&. :: ii.zik W, IS 0 -dj~-~--.4 a' -orientation m696

w0 -04 -1 'b'-orientation 0

-.4- multiple inserts , '-, ') transfect into helper virus virus) (fowlpox . infected cells 0. infected cell for packaging 2 4 S () isolate virus, infect mammalian cells FIG. 2. Southern blot analysis ofthe modified viruses obtained by nonpermissive for helper virus for direct insertion of the gpt gene cassette into the single Sma I site of identification of chimeric vaccinia virus vaccinia virus. Total DNAs of infected cells were isolated, digested with HindIll, electrophoresed in a 0.7% agarose gel, and further mpoxvirus promoter = foreign gene vaccinia genome processed for Southern blotting. The blot was hybridized to a 32P-labeled gpt probe (pSV2gpt; ATCC 37145). Lanes 1-8, DNAs FIG. 1. Construction of chimeric vaccinia viruses by molecular derived from the cells infected with the plaques F12.2-F12.9; lanes cloning and in vivo packaging. The vaccinia virus genome (WR 9-13, DNAs from cells infected with the plaques F13.1-F13.5; lane strain) contains a single Sma I restriction endonuclease cleavage site. 14, DNA from CV-1 cells infected with wild-type (wt) vaccinia virus; Viral genomic DNA was cleaved at this single site and ligated with lane 15, DNA from noninfected CV-1 cells; lane 16, size markers DNA fragments of a model gene cassette. The ligated DNA was (HindIII fragments of phage A DNA). Arrows at right indicate the transfected into fowlpox helper virus-infected avian cells for pack- sizes of the markers in kilobase pairs. Viral isolate F12.9 (lane 8) did aging. Vaccinia chimeras with single and multiple inserts were not grow. The isolates in lanes 5 and 9 were not compatible with any obtained. Control transfections of the ligated DNA into noninfected of the structures predicted in Fig. 3. They may therefore be illegit- avian cells did not result in virus progeny. imate structures and were not further analyzed. Downloaded by guest on September 26, 2021 Biochemistry: Scheiflinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9979 Chimeric Viruses with Inserts in Both Orientations Are F-IlindIIl fl \c I -- Formed. Thirteen Gpt+ vaccinia virus plaques were isolated from CV-1 cells and amplified under Gpt selection (15, 20). trf 11- " J-rt1r-- 4 Total DNA of infected CV-1 cells was digested with the -''l~rN.N'N -4 restriction endonuclease HindIII and subjected to Southern blotting. A single hybridizing band of45.4 kb was detected in four cases (Fig. 2, lanes 2, 4, 11, and 13), confirming the insertion of one copy of the cassette into the viral genome in the b orientation (Fig. 3). The a orientation shown by a single I-A.4 gpt-hybridizing fragment of5.7 kb, was obtained in two cases (Fig. 2, lanes 7 and 12). The patterns of three viral isolates I-_.)..j were consistent with a tandem a configuration (Fig. 2, lanes 1, 6, and 10); in these cases fragments of 5.7 and 1.1 kb hybridizing with the gpt probe would be expected (Fig. 3). One isolate had a tandem b structure as indicated by frag- -1 2.. ments of 45.4 and 1.1 kb hybridizing with the gpt probe (Fig. 2, lane 3). This experiment demonstrated that the direct -.4-1.4 cloning approach resulted in a variety of genomic configura- -41I. tions of the chimeric viruses. To further investigate the various genomic structures, the |4 0j), viruses F13.4 (a orientation), F12.5 (b orientation), F12.7 and I 2 3 4 {1.1|11 1 F13.2 (tandem a orientation), and F12.4 (tandem b orienta- tion) were plaque-purified under Gpt selection three times, FIG. 4. Restriction analysis ofthe chimeric vaccinia viruses. The amplified without selection, and grown to large scale. Viral DNAs were digested with HindIII or Nco I and electrophoresed in to Southern analysis. The an agarose gel. The photograph shows an ethidium bromide-stained DNA was prepared and subjected gel of HindIII-digested DNAs of the viruses F13.4, F12.5, F12.7, HindIII patterns of the purified viruses were confirmed. In F13.2, and F12.4 and of wild-type virus (lanes 1-6); Nco I digests of the single-insert viruses, extra bands of 5.7 and 5.2 kb the same viruses (lanes 8-13); and, as size markers, A HindIII and (indicating the a and the b orientation, respectively) were 4X174 Hae III fragments (lanes 7 and 14, respectively). Arrows at present (Fig. 4, lanes 1 and 2), and the 5.7- or the 45.4-kb right indicate sizes ofmarkers in kilobase pairs. Orientation [a, b, or fragment contained the hybridizing sequences (Fig. 5, lanes tandem a or b (ta or tb)] of the inserted DNA is indicated for each 1 and 2). In the viruses with tandem insertions, the 5.7- and chimeric virus. 1.1-kb fragments, indicating tandem a structure, and the 45.4- and 1.1-kb fragments, indicating tandem b structure, hybrid- predicted Nco I patterns, the viruses with the tandem inserts ized with the gpt probe (Fig. 5, lanes 3-5), confirming their had surprising structures. A single insertion into the 0.6-kb expected structures. wild-type Nco I fragment, which contains the unique Sma I Formation of Families of Chimeric Viruses with One to site, resulted in a new 1.7-kb fragment hybridizing with the Multiple Inserts. While the single-insert viruses showed the gpt probe (Fig. 5, lanes 9 and 10). The additional bands in the 9- to 15-kb size range were probably contaminants caused by H S H Hind MA small viral subpopulations generated by occasional internal I & I I fragment recombination events of the 300-base-pair P7.5 promoter 44.6 4.9 region of the inserted P7.5 promoter/gpt gene cassette and H H H single a- I &y d:M I orientation ____ HinId IllI i -~ \t'! |i "f 44.9 5.7 H H H single 'b'- - I. - - 7r l] r .-. -- orientation N- N N N ! A 4 -4_ an -N 4 -i,! -l _t- 7 - 7 45.4 5.2 _ ,'7 r_, F 7 _ __ _ n As _~~~~ ~ ~ ~ ~ ~~~~~~~. -An H H H H tandem a- _o 1 I I 4 ,_ I,_ orientation 44.9 .1 5.7 u. 40.. n H H H H H tandem b'- -00- 1 orientation 45.4 .1 5.2

FIG. 3. Predicted structures ofthe modified HindIII A fragments. Single and tandem insertions (multimers) of the model gene cassette into the unique Sma I site are shown. The 1.1-kb cassette is composed of a 300-base-pair promoter segment separated by a HindIII site from the 800-base-pair gpt gene. HindI11 (H) and Sma I (S) restriction endonuclease cleavage sites are indicated. Numbers 1 2 I . 8 U'.) a. 2i.. - indicate the sizes of the respective fragments in kilobase pairs. The sizes of the HindIlI fragments that hybridized with the gpt probe are FIG. 5. Autoradiograph of a Southern blot (same DNA samples given in boldface type; arrows indicate the direction of transcription as in Fig. 4) hybridized to the gpt probe. A strong overexposure of of the gene cassette. Head-to-head or tail-to-tail structures (which the Nco I-digested samples is shown to make the higher multimers would result in either a 45.4- and a 5.7-kb or a single 1.6-kb clearly visible. The weak additional bands in the 9- and 15-kb range gpt-hybridizing fragment, respectively) have not been observed. (lanes 9 and 10) are probably contaminants (see text). Downloaded by guest on September 26, 2021 9980 Biochemistry: Scheiflinger et al. Proc. Natl. Acad Sci. USA 89 (1992) the corresponding promoter regions of the two endogenous bridization with a fowlpox virus probe prepared from sucrose 7.5-kDa-protein genes (21). A tandem double insertion would gradient-purified virions. No cross-hybridization of the vac- result in a single 2.8-kb gpt-hybridizing Nco I fragment. The cinia viruses with fowlpox virus DNA was observed (data not analysis of the tandem-insert viruses, however, revealed a shown). ladder of gpt-hybridizing fragments starting with the 1.7-kb monomer up to 1O-mers and differing in increments of 1.1 kb DISCUSSION (Fig. 5, lanes 11-13). In summary, the Southern analysis of the purified viruses showed that both viruses with single' The insertion of foreign DNA into unique restriction endo- insertions in different orientations had the predicted nuclease cleavage sites of the 200-kb vaccinia virus genome and that tandem insertions in either orientation gave rise to by molecular cloning has been demonstrated. Viable chi- recombination events leading to multimerization of the in- meric virus was obtained by packaging in fowipox helper serted foreign genes. virus-infected host cells. Four types of viruses were isolated: Vaccinia DNA Can Be Packaged in Mammalian Cells Abor- chimeras with single inserts in either of the two different tively Infected with Fowipox Virus. It has been shown previ- orientations and chimeras with multiple tandem inserts in ously that fowlpox virus can also infect mammalian cells; both orientations. The formation of families of recombinants however, the viral cycle is not completed in these with multiple inserts was an unexpected result. It can be best non-typic host cells. Depending on the cell type, viral growth explained by the hypothesis that a chimeric virus with more stops in either the early or the late stage and viable fowlpox than one insert in tandem orientation, which consists of one virus is not formed (22). These findings prompted us to defined genomic DNA molecule at the prereplication stage, investigate packaging vaccinia DNA in a continuous mam- forms a family of chimeric viruses with one to multiple inserts malian cell line. Confluent monolayers of CV-1 cells were during further replication cycles. However, due to growth infected with fowlpox virus strain HP1.441 at 0.5 pfu per cell advantages, the chimeras with single inserts seem to be the and then transfected with a ligation mixture consisting ofNot most abundant subpopulation. A similar effect has been I-cleaved vaccinia virus DNA and a gpt gene cassette having found after conventional insertion of triplicated DNA seg- Not I flanking sites. The single Not I site in vaccinia virus is ments into vaccinia virus (23). The conventional insertion of located in an intergenic region in the HindIll F fragment (19). two envelope genes from different human immunodeficiency After incubation for 3 days, the cells were harvested and the virus strains has been used to generate hybrid envelope crude viral stock was titered on CV-1 cells in the presence or proteins (24). The generation of families of chimeras after absence of Gpt-selective medium. The outcome is summa- tandem insertions of the highly homologous genes may, in rized in Table 1. The most important result was that fowlpox addition, serve to generate hybrid proteins more easily. virus could package the modified vaccinia DNA in a cell type Avi- and orthopoxviruses belong to different genera and that prevents its own growth. Moreover, the yield ofchimeric have different host ranges. Since a cell line that did not plaques was in the range 5-10%. This compares favorably support the growth ofthe helper virus was used for the plaque with the in vivo recombination technique, in which usually purifications ofthe packaged virus, all plaques obtained were about 0.1% ofthe total plaques are recombinants. Ligation of derived from packaged vaccinia viruses. The packaging pro- the vector arms alone (Table 1, experiment 5) resulted in a cedure of vaccinia genomes by fowlpox helper virus is higher titer than ligation experiments 1-4, probably due to therefore followed by a powerful selection step that results in lack of contaminants present in the agarose-purified insert the complete removal of the helper virus and the exclusive molecules. growth of the packaged virus. A similar selection procedure Some of the isolated viruses were plaque-purified and based on change of the host range has been described (25). further characterized. They showed the typical HindIII re- The packaging efficiency in avian cells of the ligated striction patterns of vaccinia virus and, in addition, foreign material from the Sma I cloning experiment using the heter- gene bands characteristic for the two possible orientations of ologous helper virus was relatively low (in the range of 5-9 the single insert. In the case of insertion into the Not I site, x 102 pfu per 6 x 106 cells). This may have been due to no viruses with multiple inserts were observed (data not shortcomings in the cloning and ligation procedure. In the shown). cloning experiments, the vector arms were not dephosphor- Heterologous Packaged Chimeric Vaccinia Viruses Do Not ylated, and side reactions, such as the ligation of the vector Cross-Hybridize with Fowlpox Virus. To study the effects of arms alone, were not excluded. Dephosphorylation of the heterologous packing by fowlpox virus on the structure ofthe vector arms, however, did not improve the yields (F.S., chimeric vaccinia viruses, the isolates F13.4, F12.5, F12.7, unpublished results). This was due to partial degradation of F13.2, and F12.4, together with four purified isolates from the the very large vaccinia vector arms (160 and 40 kb, respec- Not I cloning experiment and the fowlpox virus controls, tively) when, despite careful handling, too many manipula- were digested with HindIll and analyzed by Southern hy- tions were performed. The construction of optimized host virus strains (e.g., strains that enable forced cloning) will Table 1. Titers after abortive packaging make the ligation procedure more efficient and, in addition, Titer, pfu x 10-2 eliminate the need for a second chemical selection procedure per 6 x 106 cells (such as Gpt selection). Other factors that influence the packaging efficiency (besides obvious ones like integrity of Exp. Insert, ng - MPA + MPA Chimeras, % the arms, efficiencies of transfection and condi- 1 200 17.2 1.6 9.3 tions of cell culture) are interference phenomena at the 2 200 42.5 5.1 12.3 cellular level of the helper and the packaged virus. Under the 3 400 64.0 3.8 5.9 packaging conditions in avian cells, the fowlpox helper virus 4 400 26.8 3.8 14.2 overgrows the packaged vaccinia virus within 3 days of 5 210.0 incubation. This large excess offowlpox virus seems to cause negative interference phenomena and inhibits the growth of Vaccinia DNA (1 ,ug) was digested with Not I and ligated with the amounts of the insert DNA the packaged vaccinia virus. indicated (the P7.5/gpt gene cassette). an in some Packaging of the modified vaccinia DNA was performed in CV-1 Fowlpox virus initiates abortive infection cells abortively infected with fowlpox virus. The crude viral stock mammalian cell lines (22). Heterologous packaging of vac- was titered on CV-1 cells in the presence or the absence of Gpt cinia DNA in mammalian cells resulted in minimal cytopathic selection (mycophenolic acid, MPA). effects ofthe helper virus and resulted in about 10-fold higher Downloaded by guest on September 26, 2021 Biochemistry: Scheiflinger et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9981 yields of packaged virus compared with those obtained by 1. Moss, B. (1990) Annu. Rev. Biochem. 59, 661-688. heterologous packaging in avian cells. Transfection of mod- 2. Fenner, F., Wittek, R. & Dumbell, K. R. (1989) The Orthopox- ified genomic vaccinia DNA into fowlpox virus-infected viruses (Academic, San Diego). mammalian (CV-1) cells is therefore the method ofchoice for 3. Panicali, D. & Paoletti, E. (1982) Proc. Natl. Acad. Sci. USA 79, 4927-4931. in vivo packaging. 4. Mackett, M., Smith, G. L. & Moss, B. (1982) Proc. Natl. Acad. Avi- and orthopoxviruses do not cross-hybridize under Sci. USA 79, 7415-7419. standard conditions. Homologous recombination of the ge- 5. Ink, B. S. & Pickup, D. J. (1989) J. Virol. 63, 4632-4644. nomes of avi- and orthopoxvirus is therefore an improbable 6. Berry, G. P. & Dedrick, H. M. (1936) J. Bacteriol. 31, 50-51. event and is unlikely to occur during heterologous packaging. 7. Joklik, W. K., Woodroofe, G. M., Holmes, I. H. & Fenner, F. The generation of chimeric vaccinia virus by molecular (1960) Virology 11, 168-184. cloning and heterologous packaging is therefore independent 8. Fenner, F. & Woodroofe, G. M. (1960) Virology 11, 185-201. of any recombination events between the in vitro modified 9. Joklik, W. K., Holmes, I. H. & Briggs, M. J. (1960) Virology DNA and the genome of the helper virus. In the chimeric 11, 202-218. 10. Sam, C. K. & Dumbell, K. R. (1981) Ann. Inst. Pasteur Virol. vaccinia viruses described here, no novel acquired fowlpox 132, 135-150. virus-specific genes were detected. The cross-hybridization 11. Mayr, A. & Malicki, K. (1966) Zentralbl. Veterinaermed. Reihe experiment does not exclude acquisition of fowlpox virus B 13, 1-12. genes in general but does show that recombination of this 12. Joklik, W. K. (1962) Virology 18, 9-18. kind is an infrequent event. 13. Gross-Bellard, M., Oudet, P. & Chambon, P. (1973) Eur. J. In summary, our approach allows the direct insertion of Biochem. 36, 32-38. foreign DNA into vaccinia vectors without the need for 14. Bostock, C. J. (1988) Nucleic Acids Res. 16, 4239-4252. plasmids having flanking regions homologous to viral non- 15. Falkner, F. G. & Moss, B. (1988) J. Virol. 62, 1849-1854. essential regions. Chimeric viruses with different genomic 16. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, 456-467. 17. Mackett, M., Smith, G. L. & Moss, B. (1985) in DNA Cloning: structures have been identified that, in part, cannot be A Practical Approach, ed. Glover, D. M. (IRL, Oxford), pp. obtained by the classical method. The procedure will allow 191-211. the cloning of very large DNA fragments without prior 18. Cochran, M. A., Puckett, C. & Moss, B. (1985) J. Virol. 54, subcloning in and of fragments that, due to toxic 30-37. effects, cannot be cloned in prokaryotic systems. The differ- 19. Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., ent genomic configurations of the chimeric viruses will pro- Winslow, J. P. & Paoletti, E. (1990) Virology 179, 247-266. vide a broader basis for screening of optimal viruses. Direct 20. Boyle, D. B. & Coupar, B. E. H. (1988) Gene 65, 123-128. cloning of DNA fragments downstream of endogenous viral 21. Venkatesan, S., Baroudy, B. M. & Moss, B. (1981) Cell 25, promoters may be useful for the generation of eukaryotic 805-813. 22. Taylor, J. R., Weinberg, R., Languet, B., Desmettre, P. & expression libraries. Direct cloning and packaging will also be Paoletti, E. (1988) Vaccine 6, 497-503. applicable for the construction of chimeric poxviruses of 23. Hruby, D. E., Schneewind, O., Wilson, E. M. & Fischetti, other genera and should therefore be a powerful alternative V. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3190-3194. to the classical in vivo recombination approach. 24. Gritz, L., Destree, A., Cormier, N., Day, E., Stallard, V., Caiazzo, T., Mazzara, G. & Panicali, D. (1990) J. Virol. 64, We thank G. Gerstenbauer for expert technical assistance, Dr. A. 5948-5957. Mayr for providing the fowlpox virus strain HP1.441, and Drs. C. 25. Perkus, M. E., Limbach, K. & Paoletti, E. (1989) J. Virol. 63, Gibbs and N. Barrett for critically reading the manuscript. 3829-3836. Downloaded by guest on September 26, 2021