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

Abstract

The distribution and orientation of origin-binding protein (OBP) sites are the main architectural contrasts between varicellazoster virus (VZV) and herpes simplex virus (HSV) origins of DNA replication (oriS). One important difference is the absence of a downstream OBP site in VZV, raising the possibility that an alternative cis element may replace its function. Our previous work established that Sp1, Sp3, and YY1 bind to specific sites within the downstream region of VZV oriS; we hypothesize that one or both of these sites may be the alternative cis element(s). Here, we show that the mutation of the Sp1/Sp3 site decreases DNA replication and transcription from the adjacent ORF62 and ORF63 promoters following superinfection with VZV. In contrast, in the absence of DNA replication or in transfection experiments with ORF62, only ORF63 transcription is affected. YY1 site mutations had no significant effect on either process. Recombinant viruses containing these mutations were then constructed. The Sp1/Sp3 site mutant exhibited a significant decrease in virus growth in MeWo cells and in human skin xenografts, while the YY1 site mutant virus grew as well as the wild type in MeWo cells, even showing a late increase in VZV replication in skin xenografts following infection. These results suggest that the Sp1/Sp3 site plays an important role in both VZV origin-dependent DNA replication and ORF62 and ORF63 transcription and that, in contrast to HSV, these events are linked during virus replication.V aricella-zoster virus (VZV) is a ubiquitous human alphaherpesvirus and is the causative agent of two diseases, varicella (chickenpox) and herpes zoster (shingles). The VZV genome (125 kb) contains at least 71 genes and contains two origins of DNA replication (oriS) flanked by the ORF62 and ORF63 genes (7). VZV oriS contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin-binding protein (OBP) (ORF51). All three OBP-binding sites (boxes A, B, and C) for the VZV OBP [5=-C(G/A)TTCGCACT-3=] are upstream of the palindrome and in the same orientation (Fig. 1). This is in contrast to the structure of herpes simplex virus (HSV) oriS, where the OBPbinding sites (boxes I, II, and III) are located both upstream and downstream of the AT-rich element. These binding sites are also oriented in different directions and use different DNA strands (42).Stow et al. (43) showed previously that the A site is absolutely required for DNA replication and that the deletion of the C site results in a decreased level of replication in VZV. In contrast, the deletion of the B site showed no effect on the extent of replication. Thus, the minimal VZV origin consists of the A site and the ATrich palindrome. In contrast, all three OBP-binding sites in HSV are required for efficient DNA replication (1,9,13,23,31,41,46). These data suggest that the OPB binding pattern, the unwinding of the origin, and the development of the replication fork may be different in VZV and HSV.One possibility that might resolve this apparent differenc...

Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein

“…Mutation of their binding sites not only inhibited binding but also suppressed VZV origin-dependent DNA replication in DpnI assays (Khalil et al, 2012). We performed DpnI replication assays in the presence of ectopically expressed Sp family members in order to further examine the influence of these cellular transcription factors on VZV DNA replication.…”

“…We performed DpnI replication assays in the presence of ectopically expressed Sp family members in order to further examine the influence of these cellular transcription factors on VZV DNA replication. These experiments involved co-transfection of the pLitmus R62/63F plasmid (Khalil et al, 2011 and Khalil et al, 2012) containing the VZV oriS sequence, with individual plasmids expressing each of the Sp factors into MeWo cells, followed by VZV superinfection at a ratio of 0.4/1 infected to uninfected cells.…”

“…We know from our prior work that mutation of the Sp1/Sp3 site in the downstream region of VZV oriS inhibits oriS-dependent DNA replication in MeWo cells (Khalil et al, 2012). …”

“…Recently we reported that Sp3, in addition to Sp1, binds to the downstream region of VZV oriS and to the ORF3 promoter. Mutation of their binding sites reduced ORF62, ORF63 and ORF3 expression in reporter gene assays (Khalil et al, 2012; and Khalil et al, 2013). The contributions of Sp2 and Sp4 to VZV gene expression and replication remain unknown.…”

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

“…ORF3, gI, gE, ORF28/29, ORF10) and the downstream region of oriS were reported previously (Ito et al, 2003; Peng et al, 2003, Yang et al, 2004; Berarducci et al, 2007; Che et al, 2007; Khalil et al, 2008, and Khalil et al, 2013). Mutation of their DNA binding sites on VZV promoters inhibited the activation of these VZV promoters, suggesting that their interaction with promoters (and IE62) is important for efficient VZV replication (Khalil et al, 2012; and Khalil et al, 2013). …”