Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication

Abstract

The varicella-zoster virus (VZV) origin of DNA replication (oriS) contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin binding protein (OBP) encoded by VZV ORF51. All three OBP binding sites are upstream of the palindrome in contrast to the sequence of the herpes simplex virus oriS, which has required OBP binding sites upstream and downstream of the AT-rich region. We are investigating the roles that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes, GC box 1 and GC box 2, in the downstream region which were predicted to be binding sites for the cellular transcription factor Sp1. Electrophoretic mobility shift assay and supershift assays showed that two members of the Sp family (Sp1 and Sp3) stably bind to GC box 1, but not to GC box 2. A predicted binding site for the cellular factor Yin Yang 1 (YY1) that overlaps with GC box 2 was also identified. Supershift and mutational analyses confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding and an increase in origin-dependent replication efficiency in DpnI replication assays. In contrast, mutation of the YY1 site had a statistically insignificant effect. These results suggest a model where origin-dependent DNA replication and viral transcription are coupled by the binding of Sp1 and Sp3 to the downstream region of the VZV replication origin during lytic infection. They may also have implications regarding establishment or reactivation of viral latency.Varicella-zoster virus (VZV) is a member of the Alphaherpesvirus subfamily of the Herpesviridae family. VZV is the causative agent of two types of disease, chicken pox (varicella) upon primary infection and shingles (zoster) after reactivation from latency (1, 2). The VZV genome consists of 125 kbp which encodes approximately 69 open reading frames (ORFs). The viral genome is made up of long and short unique segments designated U L and U S, respectively, both of which are bounded by inverted repeat sequences (11). The linear sequence of VZV genes is similar to that of herpes simplex virus type 1 (HSV-1) and includes the coding sequences for orthologues of the seven HSV-1 proteins required for origin-dependent DNA replication (9).The VZV genome contains two origins of DNA replication (oriS) within the internal repeats (IRs) and terminal repeats (TRs) bounding the U S segment at sites analogous to those within the HSV-1 genome. VZV, however, lacks the third HSV-1 origin (oriL), which is located near the center of the HSV-1 U L region (10,11,48,49). HSV-1 oriL has been shown to be dispensable for replication of HSV DNA (3, 53) but has been implicated in HSV pathogenesis and reactivation from latency (4). The equivalent region in the VZV genome is comprised of a bidirectional promoter that regulates the transcription of the VZV DNA polymerase catalytic subunit (ORF28) and DNA binding protein (ORF29) genes (32, 54).The architecture of the VZV and HSV-1 oriS regions ...

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 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).…”

“…Sp1 was shown previously to interact with the VZV major transcription activator IE62 (28,32), and there is a high incidence of predicted Sp1-binding sites within VZV promoters (37). The role of other Sp family members in VZV infection and replication is still largely unknown, but the demonstration of Sp3 binding to the Sp1/Sp3 site in the downstream region of oriS provides evidence for a role for this transcription factor in the VZV life cycle (16).…”

“…The VZV OBP does not bind to this sequence (5,43), but the plasmids used in the original identification of the minimal VZV oriS contained this 125-bp downstream sequence, which included the Sp1/Sp3 and YY1 sites. We therefore wished to determine if the Sp1/Sp3-and YY1-binding sites that we studied previously (16) were involved in origin-dependent DNA replication and/or in the expressions of the ORF62 and ORF63 genes, which flank VZV oriS.…”

“…In previous work (16), we identified the binding of the cellular transcription factors Sp1, Sp3, and YY1 to portions of the downstream region of VZV oriS. YY1 appeared to have no effect on origin-dependent DNA replication.…”

“…Three hours before transfection, the medium was replaced with fresh medium. Origindependent DNA replication experiments were performed as described previously by Stow and McMonagle (41), Stow and Davison (42), and Khalil et al (16). The cells were transfected with 5 g of wild-type or mutant pLitmus R62/63F plasmids.…”