**2.2 Construction of the VZV BAC (VZVBAC)**

Generally, BAC vectors can be directly inserted into viral genomes via homologous recombination. However, this method cannot be used to construct the VZVBAC because the virus' highly cell-associated nature makes isolation of the VZV genome and purification of recombinant plaques difficult (Nagaike et al., 2004). Instead, VZVBAC clones are constructed using a set of four overlapping cosmids spanning the entire VZV genome (Fig. 2).

Fig. 2. Schematic diagram the VZV genome and four-cosmid system. The VZV clinical strain, p-Oka, consists of a 125-kb genome with unique long (UL) and unique short (US) segments. Four cosmids with overlapping VZV genomic segments are shown. The BAC vector-containing plasmid, pUSF-6, was inserted between ORF60-61.

First, the pUSF-6 vector was inserted into VZV cosmid pvSpe23, between ORF60 and ORF61 (Fig. 2), via homologous recombination (Yu et al., 2000). The BAC-containing cosmid was then co-transfected with three complementary cosmids (Niizuma et al., 2003) into human melanoma (MeWo) cells. Because each of the four cosmids contains an overlapping sequence, the cosmids can recombine into one large circular virus genome via homologous recombination to create the recombinant virus. Our use of the GFP selectable marker in the pUSF-6 vector allowed for visualization of the recombinant VZVBAC in plaques that form post-transfection (Fig. 3C). Finally, the VZVBAC DNA from the infected cells was purified and transformed into *E. coli*. Chloramphenicol-resistant colonies were selected for and used to isolate the desired VZVBAC DNA (Zhang et al., 2007).

We used restriction enzyme digestion and DNA sequencing to verify the integrity and stability of the VZVBAC DNA. VZVBAC DNA was digested by HindIII, yielding the predicted digestion pattern with a sum of ~130 kb (Fig. 3G), thus indicating that no large deletions and rearrangements were present. In addition, the ORF62/71 gene was sequenced to check for base-pair changes in the VZVBAC genome after synthesis in *E. coli*. This large duplicated gene, encoding an immediate-early transactivating protein (Perera et al, 1992), was amplified via PCR and cloned into a pGEM-T vector for sequencing. Because the ORF62/71 sequences in the VZVBAC were identical to those of the published p-Oka strain, we can conclude that the BAC DNA in *E. coli* is stable.

Fig. 3. Construction of VZV BAC. (A) The BAC-containing cosmid was co-transfected with the three complementary cosmids into MeWo cells. (B) Homologous recombination between cosmids formed a circular, full-length VZVBAC genome. (C) The recombinant BAC was replicated, and produced a plaque visualized with the GFP marker. (D) Circular DNA was isolated from infected cells, (E) transformed into *E. coli*, and selected for cmR colonies. (F) The VZVBAC DNA was isolated from *E. coli* and (G) verified by restriction digestion and partial sequencing. (H) The infectivity and integrity of the VZVBAC were tested by transfecting BAC DNA into MeWo cells to generate the VZV virus.

The GFP marker in the viral genome was also tested; MeWo cells were infected with VZVBAC and continuously passed four times (1:10 dilution) over two weeks. On examination of the plaques under a fluorescent microscope, all VZVBAC-infected cells fluoresced green,

then co-transfected with three complementary cosmids (Niizuma et al., 2003) into human melanoma (MeWo) cells. Because each of the four cosmids contains an overlapping sequence, the cosmids can recombine into one large circular virus genome via homologous recombination to create the recombinant virus. Our use of the GFP selectable marker in the pUSF-6 vector allowed for visualization of the recombinant VZVBAC in plaques that form post-transfection (Fig. 3C). Finally, the VZVBAC DNA from the infected cells was purified and transformed into *E. coli*. Chloramphenicol-resistant colonies were selected for and used

We used restriction enzyme digestion and DNA sequencing to verify the integrity and stability of the VZVBAC DNA. VZVBAC DNA was digested by HindIII, yielding the predicted digestion pattern with a sum of ~130 kb (Fig. 3G), thus indicating that no large deletions and rearrangements were present. In addition, the ORF62/71 gene was sequenced to check for base-pair changes in the VZVBAC genome after synthesis in *E. coli*. This large duplicated gene, encoding an immediate-early transactivating protein (Perera et al, 1992), was amplified via PCR and cloned into a pGEM-T vector for sequencing. Because the ORF62/71 sequences in the VZVBAC were identical to those of the published p-Oka strain, we can

Fig. 3. Construction of VZV BAC. (A) The BAC-containing cosmid was co-transfected with the three complementary cosmids into MeWo cells. (B) Homologous recombination between cosmids formed a circular, full-length VZVBAC genome. (C) The recombinant BAC was replicated, and produced a plaque visualized with the GFP marker. (D) Circular DNA was isolated from infected cells, (E) transformed into *E. coli*, and selected for cmR colonies. (F) The VZVBAC DNA was isolated from *E. coli* and (G) verified by restriction digestion and partial sequencing. (H) The infectivity and integrity of the VZVBAC were tested by

The GFP marker in the viral genome was also tested; MeWo cells were infected with VZVBAC and continuously passed four times (1:10 dilution) over two weeks. On examination of the plaques under a fluorescent microscope, all VZVBAC-infected cells fluoresced green,

transfecting BAC DNA into MeWo cells to generate the VZV virus.

to isolate the desired VZVBAC DNA (Zhang et al., 2007).

conclude that the BAC DNA in *E. coli* is stable.

signifying the stability of the GFP marker in the viral genome. Lastly, the infectivity and integrity of the VZVBAC were confirmed by transfecting BAC DNA into MeWo cells to produce the virus. A summary of the process to construct a VZV BAC and verify its integrity is illustrated in Fig. 3.
