**5.1 Essential VZV ORFs**

72 Bacterial Artificial Chromosomes

mini-preparation of BAC DNA to confirm the ORF deletion and kanR replacement by PCR. Lastly the PCR-verified clones were chosen for maxi-preparation of BAC DNA and digested with HindIII to ensure that only the targeted sequence was deleted. When the digestion pattern of the deletion clone was compared to that of the parental WT VZVLuc clone, no

Finally, to generate VZV deletion mutant viruses, these verified clones were transfected into MeWo cells, along with WT VZVLuc DNA. The size and growth kinetics of the virus as measured by resultant plaques, or absence of plaques, are indicative of the essentiality of a

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

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

additional deletions from the genome were detectable (as shown in Fig. 3G).

particular VZV ORF for viral replication, discussed later.

phenotypes should be fully restored in these rescue viruses.

**4.3 Generation of a VZV rescue clone** 

The results indicate that among VZV's 70 unique ORFs, 44 ORFs are essential for viral replication in cultured MeWo cells, while 26 ORFs are nonessential (Zhang et al., 2010). Fig. 8 provides a visual representation of the entire VZV genome and categorizes the essentiality of each ORF based on the growth properties of its corresponding deletion mutant virus.

Fig. 8. VZV genome-wide functional profiling based on analysis of single viral ORF deletion mutants. Each VZV ORF is color-coded according to the growth properties of its corresponding virus gene-deletion mutant in cultured MeWo cells and human fetal skin organ cultures. The grey lines for ORF42 represent a splicing junction. For all growth curves, wildtype infections served as positive controls and mock infections served as negative controls.

Various studies, cumulatively, have found that the essential VZV ORFs encode genes for viral structural proteins, transcriptional regulatory proteins, and enzymes involved in DNA replication. The majority of these crucial ORFs encode proteins with imperative functions in maintaining the viral life cycle. For example, some ORFs are a part of the viral tegument and encode immediate-early proteins with transcriptional regulatory activity (Perera et al., 1992; Defechereux et al., 1993; Moriuchi et al., 1994). Other ORFs encode phosphoproteins primarily contained in the nuclei of infected cells (Moriuchi et al., 1993). It has also been reported that most of the VZV ORFs encoding glycoproteins also belong in this group of genes indispensable for viral replication (Mallory et al., 1998; Yamagishi et al., 2008).

Upon further analysis, we found that essential VZV genes have significantly different enrichment for functional categories than nonessential genes. As depicted by the distribution of functional annotations (Fig. 9A), essential VZV genes are significantly enriched for DNA replication and DNA packaging. These include genes encoding the subunits of VZV DNA polymerases, DNA binding proteins, DNA packaging proteins, and nucleocapsid proteins.
