Transcription Factor YY1 Represses Cell-Free Replication from Human Papillomavirus Origins

The small double-stranded DNA tumor viruses simian virus 40 (SV40), polyomavirus, and papillomaviruses are model systems for studying eukaryotic DNA replication because they rely heavily on host proteins. Many cellular replication proteins have been identified and cloned based on cell-free replication initiated by the SV40 large T antigen (reviewed in reference 53). Human papillomaviruses (HPVs) are widespread pathogens that infect various epithelia, causing warts (reviewed in reference 65). Infections by high-risk HPVs, including HPV type 18 (HPV-18), can develop into dysplasias and cancers in which the viral DNA often becomes integrated into the host chromosomes, an event proposed to play an important role in disease progression (reviewed in reference 10). In productively infected benign lesions, two distinct modes of DNA replication make this virus family attractive as a possible paradigm for regulated host DNA replication. In these lesions, HPV DNA is maintained as low-copy-number, extrachromosomal plasmids in the basal and parabasal cells of the squamous epithelium. Only in the differentiated upper strata does vegetative amplification take place. Viral transcription also increases dramatically with cellular differentiation. These two aspects of the infection process are undoubtedly interconnected but are not yet fully understood. Because cloned genomic HPV DNA replicates poorly, if at all, in transfected cells, two strategies have been used to examine the requirements for viral origin (ori)-specific replication (reviewed in references 9 and 52). One involves transient replication of ori-containing plasmids in transfected cells in the presence of vectors expressing viral replication proteins. E1 and E2 proteins from the same viral type and certain mixed pairs from distinct types can support replication from homologous and heterologous papillomavirus ori’s with varied efficiencies (8, 12, 18, 55, 56, 59, 64). The second assay is cell-free replication, which has been established for HPV-11 and bovine papillomavirus type 1 (BPV-1), using cellular extracts supplemented with viral proteins purified from insect Sf9 cells infected with recombinant baculoviruses (23, 61). These studies show that both the viral proteins and their ori’s are highly conserved among HPVs and animal papillomaviruses. Efficient ori replication requires both E1 and E2 proteins, with few exceptions (3, 18). The functions of the papillomavirus replication proteins have been examined extensively. Briefly, the HPV-11 and the BPV-1 E1 protein binds to an imperfect palindrome (the E1 binding site [E1BS]), which is flanked by E2BSs (Fig. ​(Fig.1)1) (30, 47, 54, 60, 61). The E1 protein is an ATPase and helicase (5, 20, 31, 49, 62) and also binds to the 180-kDa catalytic subunit (4, 41) and p70 subunit (11) of the host DNA polymerase α. In cell-free replication, HPV-11 and BPV-1 E1 are required throughout initiation and elongation, as expected of a helicase at the replication fork (30). The E1 proteins also exhibit relatively high nonspecific DNA binding, which undoubtedly contributes to their ability to promote a low level of E2-independent as well as ori-independent replication when present at elevated concentrations in cell-free systems (23, 61, 62). The functions of the E2 proteins are severalfold. All E2 proteins are dimeric transcription factors and bind to a consensus sequence ACCN6GGT that exists in multiple copies in the upstream regulatory region (URR) of all papillomaviruses (9, 52). The binding of E1 protein to the E1BS is stabilized by interaction with the E2 proteins bound to nearby sites (17, 33, 36, 47, 48, 54). At low E1 concentrations in the cell-free system, the assembly of initiation complex on the ori requires both E1 and E2 proteins (30). These interactions provide specificity and increase the efficiency for ori replication (7, 23, 36, 46). However, the E2 protein appears to be dispensable for elongation (30). FIG. 1 Replication origins of HPV-18 and HPV-11. Indicated are the binding sites for viral proteins E1 and E2 and for cellular transcription factors, YY1, Sp1, and TBP. The YY1BS in HPV-18 ori overlaps the putative E1BS (1, 25). As expected from the properties of the E1 and E2 proteins, the papillomavirus ori regions are highly conserved and consist of several copies of the E2BS and one proven or putative E1BS, overlapping the transcription enhancers and E6 promoter. One or more copies of the E2BS are critical to ori activity of genital HPVs, such as HPV-11 and HPV-18, whereas deletion or mutation of E1BS reduces but does not abolish the activity (7, 23, 31, 32, 43, 56). An efficient HPV-18 ori has been localized by transient-replication assays to a 210-bp fragment spanning nucleotides (nt) 7766 to 7857 and 1 to 119 (43, 55, 56). It contains three copies of the E2BS and one putative E1BS as inferred by analogy to the HPV-11 and BPV-1 E1 protein binding sequences (Fig. ​(Fig.11). In the ori of SV40 or polyomavirus, the ori core binds the viral T antigen. In addition, transcription factor binding sites flanking the core are auxiliary elements that enhance ori activities. Enhancement of replication in vivo is attributed to the ability of bound transcription factors to prevent nucleosome formation around the ori (reviewed in reference 13). The BPV-1 E2 protein also functions in this capacity (29). In the short HPV-18 ori, there are several host transcription factor binding sites, including those for yin-yang 1 (YY1), which overlaps the EIBS, for Sp1, and for TATA-box binding protein (TBP) (Fig. ​(Fig.1).1). The Sp1 and TBP sites are positive transcriptional elements (see references 1, 14, 15, 42, and 57 and references therein), and mutations in these sites have a negative effect on transient replication (25, 45). YY1 protein interacts with diverse proteins, including TBP, transcription factor IIB, Sp1, c-Myc, and adenovirus E1A proteins (for a review, see reference 51). In some instances, YY1 and other transcription factors compete for binding to adjacent or overlapping sites. Thus, depending on the sequence context, YY1 either functions as a transcription repressor, an activator, or an initiator binding protein. It is of great interest to determine whether or how YY1 protein might regulate ori replication. In this report, we describe our findings concerning the effect of YY1 in a cell-free replication system established with human 293 cell extracts supplemented with HPV-18 E1 and E2 proteins, each expressed as a fusion protein in Escherichia coli. In this system, efficient HPV-18 ori replication is dependent on both E1 and E2 proteins. We found that YY1 inhibited HPV-18 ori replication in cell-free and in transient replication. Unexpectedly, evidence suggests that inhibition is largely mediated via interference with the E2 protein functions and it does not appear to depend on binding to the ori. We show that YY1 protein interacts with E2 protein in vitro and inhibition can be relieved partially by elevated E2 protein concentrations or by an antibody to an intact YY1 protein.