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. 1999 Aug;73(8):7056-60.
doi: 10.1128/JVI.73.8.7056-7060.1999.

Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution

M Wagner  1 , S Jonjic, U H Koszinowski, M Messerle
Affiliations

Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution

M Wagner et al. J Virol. 1999 Aug.

Abstract

Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.

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Figures

FIG. 1
FIG. 1
The recombinant MCMV MC96.73 is attenuated in vivo. Newborn BALB/c mice were infected intraperitoneally with 100 PFU of wt MCMV (21 animals; solid circles) or MC96.73 virus (14 animals; open circles). Mortality of mice was monitored for 30 days postinfection, and survival rates were determined.
FIG. 2
FIG. 2
Schema for excision of the BAC vector from the viral genome in eukaryotic cells. (A) The MCMV BAC genome has an overlength due to the BAC vector insertion. Results obtained with other DNA viruses (2, 3) suggest that there is a relatively tight constraint on the length of DNA that can be encapsidated into virions. The BAC-containing genomes probably exceed this limit and are poorly packaged. (B) Insertion of the duplicate sequence d* should lead to excision of the BAC vector since the duplicated sequences (short identical viral sequences) (hatched boxes) serve as a target site for homologous recombination. The resulting unit length genomes will be preferentially packaged. This will lead to accumulation of a virus progeny with the wt genome.
FIG. 3
FIG. 3
Construction (A) and analysis (B) of full-length MCMV BAC plasmid pSM3fr and of the MW97.01 virus genome. (A) The top line (map 1) represents the right-terminal region of the wt MCMV genome with the EcoRI (E) fragments indicated (nucleotide positions are given in parentheses). Homologous recombination in E. coli between MCMV BAC plasmid pSM3 (map 2) and recombination plasmid pObfg (map 3) leads to reinsertion of the missing MCMV sequences and to the duplication of a 527-bp sequence (duplicate sequence d*) which is already present in its authentic position to the right of the BAC vector (hatched boxes), resulting in full-length MCMV BAC plasmid pSM3fr (map 4). Excision of the BAC vector by homologous recombination after transfection of the full-length MCMV BAC plasmid pSM3fr into eukaryotic cells results in the MW97.01 virus genome (map 5), whose EcoRI map is identical to that of wt MCMV (map 1). Two silent point mutations in the inserted MCMV sequences (indicated by solid circles) allow differentiation of the MW97.01 and wt MCMV genomes. Additional restriction enzyme sites indicated are AvrII (A), HindIII (H), and XbaI (X). (B) Structural analysis of BAC plasmids pSM3 and pSM3fr (panel 1) and of wt MCMV and reconstituted MW97.01 virus genomes (panel 2). Panel 1, the ethidium bromide-stained agarose gel shows EcoRI-digested DNA of BAC plasmids pSM3 and pSM3fr. The EcoRI O (Eco O), Z (Eco Z), and b (Eco b) fragments, a 3.85-kbp fragment, and size markers are indicated. Panel 2, EcoRI restriction analysis of virus genomes isolated from virions of recombinant virus MW97.01 and wt MCMV is shown.
FIG. 4
FIG. 4
Excision of the BAC vector sequences from reconstituted virus genomes. (A) Structural organization of the viral genomes before and after vector excision. Probe p detects fragments of 11.7 kbp (before vector excision) and 3.1 kbp (after vector excision) in PmlI-digested viral DNA. The duplicated sequences are indicated as hatched boxes. (B) Ethidium bromide-stained agarose gel of PmlI-digested viral DNA isolated from cells transfected with plasmid pSM3fr (first passage) and from cells infected with reconstituted virus after the second to fifth passages. PmlI-digested DNAs of BAC plasmid pSM3fr and MW97.01 virus were used as controls for the presence of the BAC vector-containing fragment and the reconstituted wt genome fragment, respectively. (C) Southern blot analysis of the viral DNAs with probe p.
FIG. 5
FIG. 5
Biological properties of the BAC-derived recombinant virus MW97.01 in vivo. Newborn BALB/c mice were infected intraperitoneally with 100 PFU of wt MCMV (21 animals; solid circles) or MW97.01 virus (15 animals; open circles). (A) Mortality of mice was monitored for 30 days postinfection (p.i.), and survival rates were determined. (B) Virus titers in organs of five animals infected with wt MCMV or MW97.01 virus were determined 12 days p.i., and median values (horizontal bars) were calculated.
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