E is a classical initiator
E1 is a classical initiator protein and, as such, plays several roles in the initiation and catalysis of viral DNA synthesis. E1 must first recognize a specific segment of the viral Lidocaine known as the “origin of DNA replication”, or “ori” for short. For most PV types examined to date, the minimal ori sequence that can support viral DNA replication in transient assays maps to the 3′ portion of the viral long control region (LCR), upstream of the early genes, and is typically comprised of two to three E2-binding sites, a palindromic E1-binding region and an AT-rich sequence, all of which are required for optimal ori function (Lee et al., 1997, Lu et al., 1993, Raj and Stanley, 1995, Russell and Botchan, 1995, Santucci et al., 1995, Spalholz et al., 1993, Sun et al., 1996, Sverdrup and Khan, 1995, Ustav et al., 1991). As reviewed in detail below, the key step in the initiation of viral DNA replication at the ori is the assembly of E1 into its enzymatically active form, a double-hexameric helicase capable of unwinding the ori and the DNA ahead of the replication fork, in an ATP-dependent manner. E1 also engages in multiple interactions with specific host factors to orchestrate the assembly of a functional replisome needed for bi-directional replication of the viral genome. The absolute reliance of E1 on host DNA replication factors for function has contributed to making PV DNA replication a model system for the study of eukaryotic DNA synthesis. Furthermore, the central role of E1 in this process and the availability of crystal structures of the enzyme have made E1 the best-studied member of the superfamily III (SF3) of helicases (Hickman and Dyda, 2005).
Domain structure of E1 The first insights into the structure and function of E1 came from the observation that it shares sequence similarity with the initiator proteins of other DNA viruses such as the large T antigen (LT-Ag) of simian virus 40 (SV40) and of other polyomaviruses (Clertant and Seif, 1984). Four conserved regions, termed A, B, C and D, were identified in the C-terminal regions of E1 and LT-Ag (Fig. 1, lower panel), which suggested that E1 may function as an ATPase (Clertant and Seif, 1984). Subsequent studies confirmed that purified recombinant E1 displays ATPase activity and is in fact a hexameric DNA helicase with 3′ to 5′ directionality (Fouts et al., 1999, Hughes and Romanos, 1993, Jenkins et al., 1996, Raj and Stanley, 1995, Rocque et al., 2000, Santucci et al., 1995, Sedman and Stenlund, 1998, Seo et al., 1993, Sheikh et al., 2003, White et al., 2001, Yang et al., 1993). Much of our understanding of the structure and function of E1 has been gathered from the study of the prototypical bovine papillomavirus type 1 (BPV1) E1 and, more recently, of E1s from prevalent anogenital human papillomavirus types (HPV6, 11, 16, 18, 31 and 33) and the cutaneous virus HPV1. Thus, one must keep in mind that our understanding of the structure and function of E1 emanates from E1 proteins from a very limited set of viruses. Although some of the basic principles underlying the mechanisms of viral DNA unwinding and replication have likely been highly-conserved during evolution, the fine tuning and regulation of E1 activity may have evolved more rapidly to accommodate differences in the life cycles of different PV types. A significant issue that has limited the biochemical characterization of the E1 helicase to only a few PV types has been the difficulty in expressing and purifying large quantities of the protein in a recombinant form that can support cell-free DNA replication. This is in part due to the fact that E1 must assemble from monomers at the ori in order to be active for DNA replication but is typically purified as large pre-formed oligomers when overexpressed in heterologous systems such as bacteria and insect cells. A notable exception has been the BPV1 E1 protein which can be readily purified from bacteria or insect cells in monomeric form or as oligomers that are in a monomer–hexamer equilibrium; a feature that has contributed to making BPV1 E1 the preferred enzyme for biochemical studies (Bonne-Andrea et al., 1995a, Fouts et al., 1999, Melendy et al., 1995, Mohr et al., 1990, Müller et al., 1994, Sedman and Stenlund, 1998). In contrast and as mentioned above, overexpression of E1 from other PV types often results in enzyme preparations comprised mostly of oligomers rather than monomers. For example, HPV11 E1 overproduced in insect cells using a baculovirus-expression system is purified mostly as hexamers that are not easily dissociated into monomers and thus poorly active in cell-free DNA replication, despite displaying significant levels of ATPase and short-duplex DNA unwinding activities (Dixon et al., 2000, Rocque et al., 2000, White et al., 2001). Fortunately, this issue has not been insurmountable and protocols have been developed to obtain HPV11 E1 from insect cells in a replication-competent form (Kuo et al., 1994). Monomeric and active HPV11 E1 can also be obtained by expression of the protein in vitro, by coupled transcription and translation in a rabbit reticulocyte lysate, a facile approach that should be easily adaptable to the E1 proteins from other PV types (Amin et al., 2000).