A common structural theme of eukaryotic replication and transcriptional promoters is the presence of arrays of binding sites for DNA binding proteins. These sites may be close together or separated by up to hundreds of base pairs, suggesting that the DNA provides a scaffold upon which complex three dimensional nucleoprotein structures are sculpted. There are many ways in which this can occur, including the formation of DNA loops and condensed nucleoprotein particles. The binding of human papillomavirus (HPV) type 11 E2 protein to the homologous ori offers an excellent model for understanding how such DNA-protein remodeling may occur and, in turn, regulate DNA function.
HPVs are a family of small DNA viruses which infect human squamous epithelia at mucosal or cutaneous sites, causing benign hyperproliferative, warty lesions. HPV has a double-stranded (ds) circular DNA genome of approximately 7.9 kb and replicates as multi-copy nuclear plasmids. Viral DNA replication requires the viral ori
and the cellular DNA replication machinery. The virus contributes two replication factors, the ori-
binding protein E2 and the replicative DNA helicase E1.1–4
The papillomaviral protein E1 is required throughout initiation and elongation5
, as it is an ATP-dependent helicase, equivalent to the cellular replicative DNA helicase (the minichromosome maintenance complex). In contrast, the ori
-binding E2 protein is only essential for the assembly of the pre-initiation complex and is absent during the elongation phase of papillomavirus DNA replication.5
In addition, E2 is also responsible for equitable viral genome segregation into daughter cells.6–11
The HPV-11 ori
spans 103 bp and lies at the 3′ end of the upstream regulatory region (URR), a 700 bp long non-coding region which also controls transcription from the adjacent E6 promoter. The ori
is comprised of three E2 binding sites (E2BSs) flanking an array of E1 BSs where E1 assembles into a di-hexameric helicase. The three E2BSs are separated by 64 and 3 bp from one another. E2BS-2 lies immediately upstream of the E1BS whereas the E2BS-3 and E2BS-4 are located just downstream. E2BS-4 is situated 3 bp upstream of the TATA sequence of the E6 promoter. By binding to the overlapping ori
/promoter elements, E2 also functions as a transcription factor and modulates the activity of this promoter. The E1 protein is recruited to the ori
by the E2 protein.12,13
Studies of HPV-11 E1 and E2 binding to the HPV ori
using electron microscopy (EM) have shown that the cellular heat shock/chaperone proteins hsp40 and hsp70 facilitate the formation of the E1 di-hexamer at the ori
, with the release of E2.14,15
In addition, there is another E2 binding site, E2BS-1, which lies 288 bp upstream of E2BS-2 and is not required for efficient transient replication in transfected cells or for cell-free replication.3,4,16
This sequence arrangement in the HPV-11 ori
is highly conserved in many HPV genotypes.
The E2 proteins vary in size among the papillomavirus types including the bovine papillomavirus (BPV), ranging from 42–48 kDa, but their functions are highly conserved. The amino terminal half of the protein is the trans
-acting domain which interacts with the E1 helicase, recruiting it to the ori
, and with transcription factors.12,13,17
A central hinge region is poorly conserved in length and in sequence. However, for HPV-11 E2 and perhaps additional mucosotropic HPV genotypes, this region contains a nuclear localization sequence and also associates with the nuclear matrix.18
The carboxyl-terminal domain promotes E2 protein dimerization, and the dimer then binds in the major groove of the DNA at a 12-base pair palindromic sequence (5′-ACCGN4CGGT-3′).19
All papilloma virus E2 proteins recognize this 12 bp sequence although the 4 base pair spacer may vary among viruses.
The BPV ori
/promoter contains a cluster of 12 E2 binding sites, and Knight et al.20
showed that BPV E2 binds DNA as a dimer and also forms larger protein particles when two E2 binding sites are spaced close together. Using EM, loops were occasionally noted when the binding sites were artificially spaced by a larger distance. This study also implicated the N terminal domain of BPV E2 in the loop formation. In an X-ray crystallographic study21
, E2N dimers were observed with HPV-16 E2, but not with HPV-11 E222
nor with HPV-18 E2 truncated of residues 1–65.21
These studies suggested that an EM examination of the binding of HPV E2 to the natural HPV ori
might reveal multiple levels of protein-mediated folding and remodeling of the DNA central to the control of HPV replication and transcription.
In this work we have carried out such a study using purified functional HPV-E2 protein and HPV-11 ori-spanning fragments containing from one to all four of the E2 binding sites. HPV-11 was selected for this study, as the structures and functions of the E1 and E2 proteins of this virus have been examined intensively, including collaborative EM work between the two laboratories involved in this present work. The 4 binding sites provide ample opportunity for remodeling events in the ori but without the complexity of the much larger number of E2 sites found in the BPV genome. We found that the cluster of three closely spaced sites generated a trimer of dimers arranged into a disk/ring-shaped hexamer. Moreover, when the DNA fragment contained all four binding sites, frequent DNA loops were observed between the distal E2BS-1 and one of the cluster of three adjacent sites some 288 bp downstream. E2C, which contains only the protein dimerization and DNA binding domain, abolished looping, indicating the amino terminal portion of the protein is necessary for DNA looping. On the basis of these structures, we discuss the implications on the assembly and regulation of functional replication complexes.