Figure 1.
A) The infectious BAC pHJO. In the pHJO BAC clone the mini-F sequences are located within the unique AvrII site of the VZV US region. RL: repeat of the long region; UL: unique region long; RS: repeat of the short region; US: unique region short. B) Preparation of the BAC for the mini-F transposition. An aphAI-I-SceI cassette (blue) was amplified with primers bearing specific 50 bp extensions (green and black). One primer provides a 50 bp duplication (red). The generated PCR product was applied to replace the cat gene of the pBeloBAC11 vector sequences (green) from pHJO, resulting in pHJOFep. C) Synchronous mini-F transposition reaction. A mini-F transfer construct was used to insert a second pBeloBAC11 vector via Red-mediated recombination of flanking 0.7 kb homologous sequences (orange and yellow) into the target position of interest. After introduction of a double-strand break by I-SceI expression, the primary mini-F cassette was seamlessly excised by a second Red-mediated recombination of the short 50 bp duplication, resulting in a pHJO variant.
Figure 2.
Infectious BAC clones with genome-intrinsic releasable mini-F sequences.
Scheme of the generation of pHJO variants with mini-F sequences (green) releasable by intrinsic genome replication processes. Upon mini-F transposition in E. coli, the vector sequences were shifted within the BAC-cloned HJO genome (pHJO) from the unique AvrII site of the US region (reverse with respect to UL, regarding the defined VZV prototype genome organization) into ORF62, ORF71, or directly into genomic terminal junction of VZV, to generate pHJOF62, pHJOF71, or pHJOFpac, respectively. Virus reconstitution from the pHJO variants yielded vector-free progeny rHJO-F-.
Table 1.
Efficiency of the mini-F vector transposition in E. coli and the vector self-excision upon virus reconstitution.
Figure 3.
Mini-F sequence transposition into the genomic terminal junction and release upon virus reconstitution.
A) Detailed overview of the genomic transitions in pHJOFpac and rHJOpac-F-. The vector sequences (green) were released upon virus reconstitution from pHJOFpac and vector-free progeny rHJOpac-F- was generated. Lengths of both transitional SalI fragments and the distances from the SalI restriction sites to the genomic junctions are given (black dotted lines). Also, the binding sites of the Southern-blot probe (red bars) for analyzing the terminal junction and of the primers P1 and P2 (Table S1) used to generate the probe are indicated. B) PCR analysis of the mini-F sequence release upon virus reconstitution. Total DNA from MeWo cells infected with pHJOFpac-derived progeny of virus passage 1–3 was analyzed with the cat-specific primers P3 and P4 (Table S1). BAC DNA of original pHJO as well as total DNA from MeWo cells infected with HJO wild-type or uninfected cells were analyzed as controls. C) Southern-blot analysis of pHJOFpac and rHJOpac-F- in comparison to HJO, pHJO, pHJOFep. BAC DNA was prepared from E. coli harboring pHJO, pHJOFep, or pHJOFpac. Total DNA from MeWo cells infected with HJO or rHJOpac-F- was purified for examination of virus DNA. The isolated DNA was digested with SalI, separated electrophoretically on a 1% agarose gel, and blotted. Genomic junction-, vector-, or VZV sequences were detected with probes specific for viral sequences spanning the genomic transition at the ORF0 end of the UL region (termed S/L0, red bars in A), the pBeloBAC11 plasmid, or pHJO DNA, respectively. The 3 kb or 6 kb SalI fragments corresponding to the terminal or internal junction as well as the 9.5 kb SalI fragments that correspond to the terminal junction with mini-F vector in pHJOFpac as presented in A are marked. Also indicated are the 1.6 kb and 1.4 kb SalI fragments that correspond to termini of linear genomes within viral DNA as outlined in A as well as the 2.6 kb SalI fragments that correspond to the restored US unique AvrII site (arrows flanking the picture frame). D) Growth kinetics of rHJOpac-F- and HJO. Multistep growth kinetics of rHJOpac-F- compared to wild-type virus HJO in MeWo cell cultures was performed. Means and standard deviations of two independent experiments with three separate results each are given.
Figure 4.
Sequence analysis after mini-F vector release from the genomic termini.
Sequence analysis of 16 cloned PCR fragments that were generated by PCR amplification of the terminal genomic junction of rHJOpac-F- or wild-type HJO DNA. The derived sequences were aligned to the sequence of the terminal transition of the original pHJO clone (first line). The GC tracts directly at the junction are highlighted in gray.