Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

Abstract

The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCEWhile much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.H uman papillomaviruses (HPVs) are small viruses with double-stranded DNA (dsDNA) genomes that infect both outer and mucosal stratified epithelium (for a recent review see reference 1). Of the more than 180 HPV genotypes described to date, about 20% infect the anogenital tract, causing either benign or precancerous lesions (2). In addition to their well-established role in inducing cervical and other anogenital cancers, HPVs have more recently been shown to be the causative agents for the majority of oropharyngeal cancers (3, 4).Only two HPV proteins, E1 and E2, are required for replicati...

Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

“…In addition to the genome sequence and oncoproteins, the viral replication proteins E1 and E2 may differ between HPV types. For example, similar mutations in the C-terminus of the E1 protein completely abolished BPV1 replication but not HPV11 replication[ 77 ]. As none of the inhibitors blocked the E1/E2-dependent replication of the 1-kbp-long HPV origin-containing plasmid URR ( S5 Fig ), it seems that Tdp1 is not crucial for the initiation of HPV replication.…”

Experimental support for human papillomavirus genome amplification early after infectious delivery

“…However, this event has never been experimentally supported due to the lack of an efficient infection model. Most research focused on transient replication of transfected established cell lines or primary keratinocytes cultures in monolayer or 3D cultures (6,15,(26)(27)(28)(29)(30)(31)(32). These systems provided valuable information but were not suitable to study the initial events after infectious delivery of viral genome to the nucleus of primary keratinocytes.…”

“…The lack of experimental support was largely due to a lack of good infection models. Most of the present knowledge about papillomaviruses replication is based on models using transfection of human epithelial cell lines (6,15,(26)(27)(28)(29)(30) or primary human keratinocytes (31,32). We now took advantage of our recently established infection model using an ECM-to-cell transfer approach to investigate the fate of viral genome after infectious entry (33).…”

Human Papillomavirus Genome Copy Number Is Maintained by S Phase Amplification, Leakage to the Cytosol during Mitosis and Lysosomal Degradation in G1 Phase

“…So far, no clear evidence in support of licensed DNA replication has been published. Therefore, other mechanisms have been proposed: tight control of E1 and E2 protein levels -both at the transcriptional and post-transcriptional (splicing) level (1,3,4,15,16); post-translational modification of E1 and E2 protein such as phosphorylation and proteolytic processing (3,17,18); and viral repressors such as the E8^E2 protein, which has been convincingly demonstrated to restrict genome copy number (5,6,19). Our data suggest that, while these mechanisms may be involved in genome copy number control, they are not sufficient.…”