Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery of infectious virus in mammalian cells

Abstract

The ability to manipulate the vaccinia virus (VAC) genome, as a plasmid in bacteria, would greatly facilitate genetic studies and provide a powerful alternative method of making recombinant viruses. VAC, like other poxviruses, has a linear, double-stranded DNA genome with covalently closed hairpin ends that are resolved from transient head-to-head and tail-to-tail concatemers during replication in the cytoplasm of infected cells. Our strategy to construct a nearly 200,000-bp VAC-bacterial artificial chromosome (BAC) was based on circularization of head-to-tail concatemers of VAC DNA. Cells were infected with a recombinant VAC containing inserted sequences for plasmid replication and maintenance in Escherichia coli; DNA concatemer resolution was inhibited leading to formation and accumulation of head-to-tail concatemers, in addition to the usual head-to-head and tail-to-tail forms; the concatemers were circularized by homologous or Cre-loxP-mediated recombination; and E. coli were transformed with DNA from the infected cell lysates. Stable plasmids containing the entire VAC genome, with an intact concatemer junction sequence, were identified. Rescue of infectious VAC was consistently achieved by transfecting the VAC-BAC plasmids into mammalian cells that were infected with a helper nonreplicating fowlpox virus.

Fig 1.

Scheme for construction of VAC–loxP–GFP–BAC. The plasmid pMBO1374–loxP was constructed by addition of a synthetic oligonucleotide containing a loxP site between the NotI and SacII sites of pMBO1374. pMBO1374–loxP–TK_RL was derived from pMBO1374–loxP by insertion of a BamHI fragment containing the inverted halves of the TK gene, separated by a SphI site. pMBO1374–loxP–TK_RL was then cleaved with NotI and ligated to a NotI fragment containing the VAC P7.5 promoter regulating GFP to form pMBO1374–loxP–GFP–loxP–TK_RL. The latter plasmid was cleaved with SphI to form a linear DNA flanked by the left and right halves of the TK gene, which was transfected into cells that were infected with either VAC strain WR or ts21 at 37°C or 31°C, respectively, to allow homologous recombination. VAC–loxP–GFP–BAC was isolated by TK-negative selection, and plaques exhibiting green fluorescence were picked several times in succession. The recombinant virus was called WR–loxP–GFP–BAC or ts21–loxP–GFP–BAC, depending on the parental virus strain.

Fig 2.

Representation of the action of Cre on head-to-head, tail-to-tail, and head-to-tail concatemers. B and C represent the terminal HindIII fragments of the VAC genome. CC, BB, and CB represent head-to-head, tail-to-tail, and head-to-tail concatemer junctions, respectively. Because of the orientation of the loxP sites, excision, and circularization of a complete viral genome only occurs with head-to-tail junctions.

Fig 3.

Screening of bacterial colonies for VAC–BACs. After transformation of E. coli with DNA from cells infected with ts21–loxP–GFP–BAC in the presence of Cre, individual colonies were screened by hybridization to ³²P-labeled probes containing DNA from the HindIII C and B terminal fragments of the VAC genome. Colonies number 5, 13, 16, 22, 34, 51, and 59 reacted with the C probe and, except for number 16, reacted with the B probe.

Fig 4.

The terminal HindIII C and B sequences of VAC DNA are fused in the VAC–BAC. Representations of the linear VAC genome and circular VAC–BAC DNA are shown in the upper left and right, respectively. DNA from five VAC–BAC clones and that of VAC genomic DNA were digested with HindIII, resolved by agarose gel electrophoresis, transferred to a membrane and hybridized to ³²P-labeled probes containing sequences from the HindIII B or C fragment of the VAC genome. The B and C probes reacted with fragments of 30 and 22 kbp of VAC WR genomic DNA but with a 52-kbp fragment of the VAC–BACs.

Fig 5.

Detection of the concatemer junction fragment in VAC–BAC DNA. The hairpin terminus and concatemer junctions are represented in the left upper and lower portions of the figure. The sites of BstEII cleavage and the resulting size fragments are indicated. The boxes with vertical lines represent tandem repeat sequences. DNA from VAC (WR) and VAC–BAC number 5 were digested with BstEII, and the products were resolved by agarose gel electrophoresis, transferred to a membrane, and probed with ³²P-labeled DNA containing tandem repeat sequences located close to the two ends of the genome. An autoradioraph is shown on the right. m, is DNA size markers. The 1.3-kbp hairpin terminus was detected in WR DNA, and the 2.6-kbp concatemer junction in the VAC–BAC. The upper band in both the WR and #5 lanes was generated by cleavage at a distal BstEII site not shown in the diagram.

Fig 6.

Size and stability of VAC–BAC DNA. (A) DNA of VAC (WR), ts21–loxP–GFP–BAC (R) and numbered VAC–BACs were analyzed by pulsed-field gel electrophoresis. Similar bands (arrow) of approximately 200,000 bp were detected by ethidium bromide staining. Only a segment of the gel is shown. (B) The same DNA samples were digested with HindIII and analyzed by agarose gel electrophoresis. Note that the BC fragments of the VAC–BACs migrate with the A fragment. In addition, the J-size fragment of WR (lower arrow) is missing in ts21–loxP–GFP–BAC and all of the VAC–BACS because of inserted DNA. In VAC–BACs 5, 13, 34, and 51, the positions of the more slowly migrating J fragment containing BAC sequences is shown by the upper arrow. In ts21–loxP–GFP–BAC and VAC–BAC 22, the J fragment also contains the GFP gene, and therefore migrates more slowly and coincides with the next higher band.