Human papillomavirus (HPV) origin-binding protein associates with mitotic spindles to enable viral DNA partitioning

Abstract

Human papillomaviruses (HPVs) establish long-term infections in patients. The mechanism for extrachromosomal HPV DNA persistence in cycling cells is unknown. We show that HPV origincontaining plasmids partition as minichromosomes, attributable to an association of the viral origin recognition protein E2 with mitotic spindles. ␣-, ␤-, and ␥-tubulins were pulled down with a tagged E2. The N-terminal transacting and C-terminal protein dimerization͞DNA binding domains independently associated with the spindles. We suggest that this E2 property enables these viruses to establish persistence. Its implication for HPV oncogenesis is presented.F or any extrachromosomal DNA virus to establish a persistent infection in cycling host cells, the viral genome must replicate and partition into both daughter cells during division. The E2 origin (ori)-binding protein of bovine papillomavirus type 1 (BPV-1) associates with mitotic chromosomes (1-3), thus providing a mechanism for viral DNA segregation. Comparable mechanisms have been demonstrated for the Epstein-Barr virus through the Epstein-Barr virus-encoded nuclear antigen 1 protein and the Kaposi's Sarcoma virus (human herpesvirus 8) through the latency-associated nuclear antigen 1 protein (4, 5). In contrast, the mechanism by which human papillomavirus (HPV) DNA partitions during cell division has not been elucidated. In this report, we demonstrate that HPV ori-containing DNA segregates as minichromosomes by association with mitotic spindles and this association is mediated by the HPV origin recognition protein E2.HPVs are medically important pathogens that establish persistent infections in long-living basal keratinocytes. Infections typically cause benign hyperproliferation of squamous epithelia in the form of cutaneous warts, laryngeal papillomas, and anogenital condylomata. Over time, infections can become subclinical, but may reactivate during episodes of immune suppression. The HPV genome is a double-stranded, circular DNA of Ϸ7,900 bp and replicates extrachromosomally in the nucleus of infected keratinocytes. Low copy numbers of the mucosotrophic HPV DNA plasmids are maintained in the basal and parabasal cells that divide, whereas the productive phase takes place only in postmitotic, differentiated cell strata and progeny virus shed within the sloughing superficial cells (6). Thus, it is paramount that, in either latent or active infections, HPV DNA must partition into the two daughters of dividing cells for viral persistence. To support viral DNA amplification in postmitotic cells, the viral E6 and E7 proteins inactivate the host tumor suppressor proteins p53 and pRB (retinoblastoma protein), respectively, reestablishing an S-phase environment. Elevated transcription of these oncogenes is normally limited to the differentiated compartment. However, if inappropriately expressed in the basal cells, such as during repeated wounding and healing, the high-risk HPV oncoproteins can promote excessive cell cycling and host chromosome instability. Indeed, a small fraction of ...

Brd4 links chromatin targeting to HPV transcriptional silencing

“…The association between Brd4 and BPV-1 E2 with chromatin appears to be stable and persists through both mitosis and interphase (McPhillips et al 2005). Since HPV E2 does not stably associate with mitotic chromosomes and seems to anchor on mitotic spindles during mitosis (Van Tine et al 2004), it remains to be investigated whether the genomic-tethering function of BPV-1 E2 can be applied to HPV E2 with respect to their functional interactions with Brd4. Likewise, it needs to be defined whether the transcriptional silencing activity of the HPV E2-Brd4 complex can also be observed with E2 complexes isolated from animal papillomaviruses.…”

“…The full-length E2 protein is a sequence-specific DNAbinding protein implicated in the control of viral DNA replication (Stenlund 2003;Abbate et al 2004), transcription (Hou et al 2002), cell cycle progression (Hwang et al 1993), apoptosis (Blachon et al 2005), senescence (Goodwin and DiMaio 2001), and viral genome maintenance and segregation (Abroi et al 2004;Botchan 2004;McBride et al 2004;Van Tine et al 2004;You et al 2004). Like many cellular transcription factors with different functional modules, E2 contains a C-terminal DNA-binding domain linked to the N-terminal transactivation domain by a flexible hinge.…”

Clonal Selection for Transcriptionally Active Viral Oncogenes during Progression to Cancer

“…Furthermore, there is also a selection against cells that express high levels of E2 which would downregulate the promoter for the viral oncogenes (9,14,16,44,63,67). An additional impetus for silencing comes from our recent observation that the HPV E2 protein associates with the mitotic spindles to enable extrachromosomal HPV DNA plasmids to segregate as minichromosomes and to establish persistence (64). However, upon viral DNA integration, E2 might contribute to genomic instability if E2 bound to the viral origin in tandemly integrated HPV DNA creates a viro-centromere, causing chromosome breakage when it is pulled to the opposite centrosome relative to the kinetochore.…”

The Human Papillomavirus Type 8 E2 Tethering Protein Targets the Ribosomal DNA Loci of Host Mitotic Chromosomes

“…In another study, the HPV-11, HPV-16, and HPV-18 alphapapillomavirus E2 proteins were reported to be associated with the mitotic spindle (37). So, while the chromosomal target of BPV has been established, the binding partners of many human papillomaviruses remain elusive, and these viruses may interact with diverse binding partners for genome segregation.…”