**4.3 Generation of a VZV rescue clone**

VZV ORF deletion rescue clones were also generated (Fig. 7) in order to show that growth defects observed in analyses of the deletion mutants are a direct result of the deleted genes, as opposed to potential mutations in other regions of the genome. Ideally, the wild-type phenotypes should be fully restored in these rescue viruses.

Fig. 7. Generation of a VZV rescue clone. (A) ORFX was amplified by PCR from the WT VZV BAC DNA and (B) directionally cloned into plasmid pGEM-lox-zeo to form pGEM-loxzeo-ORFX. (C) Amplification of the ORFX-zeoR rescue cassette by PCR using a primer pair adding 40-bp homologies flanking ORFX. (D) The PCR product was transformed into DY380 carrying the VZVLuc ORFX deletion via electroporation. (E) Homologous recombination between upstream and downstream homologies of ORFX replaced kanR with the ORFX-zeoR rescue cassette. (F) ZeoR and BAC vector sequences were removed post-verification by cotransfecting a Cre recombinase-expressing plasmid, creating the ORFX rescue clone.

To generate ORF deletion rescue clones (ORFXR), the targeted ORF deletion was amplified from wild-type VZVLuc BAC DNA by PCR. Next, the ORFX was directionally cloned into plasmid pGEM-lox-zeo to produce pGEM-zeo-ORFX. This was then used as the template to generate the ORFX-zeoR cassette via PCR using a primer pair to add 40-bp sequences, homologous to the kanR cassette flanking ORFX. In a process similar to the homologous recombination system described earlier, the PCR product was transformed into DY380 carrying the ORFX deletion genome (Fig. 7C). The kanR cassette was replaced with the ORFX-zeoR rescue cassette by homologous recombination, thus allowing for positive selection of zeocinR colonies. Because the zeoR gene within the rescue cassette is flanked by two loxP sites, it can be removed along with the BAC vector, through Cre-mediated recombination, generating the desired ORFX rescue virus. Full recovery from the marked growth defects is expected after using this rescue method to restore the wild-type ORF.
