A Sequence within the Varicella-Zoster Virus (VZV) OriS Is a Negative Regulator of DNA Replication and Is Bound by a Protein Complex Containing the VZV ORF29 Protein

Abstract

Varicella-zoster virus (VZV), human herpesvirus 3, is a member of the Alphaherpesvirinae and is the etiologic agent of two distinct clinical syndromes in humans: chicken pox (varicella), occurring during primary infection, and shingles (zoster), occurring after reactivation from latency (12). The VZV genome is a 125-kb linear double-stranded DNA molecule and is predicted to code for at least 71 proteins. The viral DNA is made up of long and short unique segments designated U L and U S , respectively, both of which are bounded by inverted and terminal repeat sequences IR s /TR s (14). The VZV genome contains two copies of an origin of DNA replication (OriS), flanked by ORF62 and ORF63 genes, within the IR s /TR s repeats bounding the U S segment (13,14,57,58). VZV has been shown to encode homologues of all seven viral gene products required for herpes simplex virus type 1 (HSV-1) DNA replication. These include the viral DNA polymerase and its associated processivity factor, a heterotrimeric helicase/primase complex, a single-strand DNA-binding protein (SSB), and a site-specific origin-binding protein (OPB) (20). However, while the two viruses have similar genomic organizations and encode similar DNA replication factors, the architecture of the VZV and HSV-1 OriS regions differs significantly (Fig. 1A).The two VZV OriS contain an AT-rich palindrome and three 10-bp consensus binding sites [5Ј-C(G/A)TTCGCACT-3Ј] for the VZV OBP encoded by VZV ORF51 (57, 59). These binding sites, designated Boxes A, B, and C, all are located upstream of the AT-rich palindrome. They are identical (Boxes A and B) or nearly identical (Box C) to the consensus binding site for the HSV-1 U L 9 OBP, with which the VZV ORF51 OBP shares 54.8% similarity and 46.5% identity (7,30,59). In addition to these cis elements, there is a characteristic GA-rich sequence immediately downstream of the AT-rich region in the VZV origin which is not present in the HSV-1 origin (14,19).All three OBP binding sites in the VZV origin are oriented in the same direction and are present on the same strand of the viral DNA. In contrast, in the HSV-1 OriS, binding sites for the UL9 OBP (Boxes I, II, and III) occur both upstream and downstream of the AT-rich region. Boxes I and II are located upstream and downstream on opposite strands of the DNA and are oriented in opposite directions. Box III is located upstream of Box I but is oriented in the same direction and occurs on the same DNA strand as Box II. Mutational analysis has shown that efficient HSV-1 origin-dependent DNA replication requires the presence of all three UL9 binding sites as well as the central AT-rich region (3,15,22,38,45,58,61).

An Sp1/Sp3 Site in the Downstream Region of Varicella-Zoster Virus (VZV) oriS Influences Origin-Dependent DNA Replication and Flanking Gene Transcription and Is Important for VZV Replication In Vitro and in Human Skin

“…In contrast, the YY1 mutation did not significantly affect the expression levels of the two genes in the absence of PAA and had no effect in the presence of PAA. These data indicate that the Sp1/Sp3 site influences the expression levels of both the ORF62 and ORF63 genes during viral replication and confirm our previously reported finding that origin-dependent DNA replication is important for high levels of flanking gene transcription (17). The statistically significant decrease in the ORF63 expression level seen in the presence of PAA using the Sp1/Sp3 mutation also suggests that the involvement of the Sp1/Sp3 site in ORF63 expression is separate from its enhancement of origin-dependent DNA replication.…”

“…This fits our data reported previously for the downstream box D site, where we showed effects on both origin-dependent DNA rep- lication and flanking gene transcription (17). Furthermore, it appears that the influence of the Sp1/Sp3 mutation on ORF62 expression is due solely to the involvement of the Sp1/Sp3 site in origin-dependent DNA replication and that origin-dependent DNA replication is required for efficient ORF62 expression.…”

“…As presented in Fig. 2B, the CGC mutation to AAA in box A showed no replicated DNA band, indistinguishable from the negative control, as we reported previously (17).…”

“…In these experiments, we used pVO2 plasmids, which lack the promoter elements of the flanking ORF62 and ORF63 genes (8,26,27). The protocol involved the transfection of the pVO2 plasmid containing the VZV oriS sequence into MeWo cells, followed by VZV-MSP (11) superinfection, as described previously (16,17).…”

“…However, previous gel shift assays with recombinant OBP did not demonstrate an interaction (5), and this site was not protected in DNase I protection assays with recombinant OBP (43). In our previous work, we designated this sequence box D and showed that the mutation of this site resulted in increased DNA replication levels in both the presence and the absence of flanking gene expression (17). These findings essentially exclude the possibility of an alternative OBP site downstream from VZV oriS that plays the role of box II in HSV oriS.…”