**5.3 Other findings**

Despite major differences between herpesvirus genomes, all the herpesviruses studied thus far have a similar number of essential genes, but varying number of nonessential genes. For example, our study suggests that the VZV genome encodes 44 essential genes and 26 nonessential genes. The herpes simplex virus 1 (HSV-1) genome encodes 37 essential genes and 48 nonessential genes (Roizman et al., 2007). Similarly, the human cytomegalovirus (HCMV), one of the largest human DNA viruses, has a genome that encodes 45 essential genes and 118 nonessential genes (Dunn et al, 2003). Furthermore, 26 of the 44 essential VZV genes have essential homologues in HSV and 18 of the 44 have essential gene homologues in HCMV, alluding that some essential genes may perform core functions for all of these herpesviruses.

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;

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

As previously mentioned, 26 of the 70 unique VZV ORFs were deemed nonessential for VZV replication in MeWo cells (Zhang et al., 2010). Of these, 8 ORFs appeared to significantly affect viral growth. In viral growth assays, the peak signals from their corresponding plaques were at least 5-fold less than the peak signals from the WT parental strain. Furthermore, atypical morphology of virally infected cells, such as reduced plaque sizes and altered syncytia formation, were also frequently observed. Studies have shown that some of these ORFs affecting optimal growth encode the small and large subunit of ribonucleotide reductase (Heineman & Cohen, 1994) and specific phosphoproteins that are

The plaques corresponding to the remaining 18 nonessential VZV ORF deletions exhibited wild-type growth in cultured MeWo cells (Zhang et al., 2010). *In vitro* growth curve analysis for viral replication showed that these ORF deletion mutants have the same growth kinetics as their wild-type parental strain, VZVLuc. Nonessential genes, in general, are significantly

Despite major differences between herpesvirus genomes, all the herpesviruses studied thus far have a similar number of essential genes, but varying number of nonessential genes. For example, our study suggests that the VZV genome encodes 44 essential genes and 26 nonessential genes. The herpes simplex virus 1 (HSV-1) genome encodes 37 essential genes and 48 nonessential genes (Roizman et al., 2007). Similarly, the human cytomegalovirus (HCMV), one of the largest human DNA viruses, has a genome that encodes 45 essential genes and 118 nonessential genes (Dunn et al, 2003). Furthermore, 26 of the 44 essential VZV genes have essential homologues in HSV and 18 of the 44 have essential gene homologues in HCMV, alluding that some essential genes may perform

post-translationally modified by protein kinases (Reddy et al., 1998).

enriched for other and unknown functional categories (Fig. 9B).

core functions for all of these herpesviruses.

Yamagishi et al., 2008).

nucleocapsid proteins.

**5.3 Other findings** 

**5.2 Nonessential VZV ORFs** 

Another observation worth noting is the size of the essential ORFs as compared to the size of the nonessential ORFs. Essential ORFs are significantly larger in size, averaging 1250-bp, while nonessential ORFs have an average size of 970-bp. Moreover, the ten largest VZV ORFs are all essential, while of the 11 smallest VZV ORFs, eight are nonessential.

Fig. 9. Distribution of functional annotations for essential and nonessential genes. (A) Distribution of functional annotations for essential genes. Essential genes are significantly enriched for DNA replication (Bonferroni corrected p-value <10-4) and DNA packing (corrected p-value <10-4) functional categories. (B) Distribution of functional annotations for nonessential genes. Nonessential genes are significantly enriched for other (corrected pvalue <10-3) and unknown (corrected p-value <0.01) functional categories. Statistical significance was determined by a hypergeometric test.
