The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment

Abstract

In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of infection is a risk factor for cancer development and is partly achieved by the attachment of viral DNA to cellular chromatin during cell division. The HPV E2 protein plays a critical role in this tethering by binding simultaneously to the viral genome and to chromatin during mitosis. We previously showed that the cellular DNA helicase ChlR1 is required for loading of the bovine papillomavirus E2 protein onto chromatin during DNA synthesis. Here we identify a mutation in HPV16 E2 that abrogates interaction with ChlR1, and we show that ChlR1 regulates the chromatin association of HPV16 E2 and that this virus-host interaction is essential for viral episome maintenance.

Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

“…Brd4 was reported to regulate HPV DNA replication, and can both activate and repress viral transcription (27). Brd4 colocalizes with E2 to tether the viral genome to chromosomes during mitotic segregation (24), which also involves the ChlR1 and TopBP1 proteins (38)(39)(40). Because of these multiple activities, the precise role of the Brd4-E2 interaction in the viral replicative program remains uncertain.…”

The eccDNA Replicon: A heritable, extra-nuclear vehicle that enables gene amplification and glyphosate resistance inAmaranthus palmeri

“…These AT-hook gene products may interact with the A/T rich regions of the eccDNA replicon and other nuclear scaffold proteins. Furthermore, the helicase domain has recently been demonstrated to be an important regulator of the chromatin association, establishment, and maintenance of the Herpes virus (Harris et al, 2017). AP_R.00g000496 is predicted to encode a helicase motif which may also have a role in tethering of the eccDNA to nuclear chromatin.…”

Plasmid Partitioning by Human Tumor Viruses

“…It is not known how HPV might bind mitotic spindles, but both BPV1 E2 and HPV16 E2 do bind the cellular protein ChlR1, a helicase that itself binds the cohesin complex (51,52). This binding, however, appears not to mediate chromosomal tethering (51). These studies and many others show that the different E2 proteins encoded by the different papillomavirus genomes use their carboxy-terminal DNA-binding domains to bind specifically to their URRs (Fig.…”

“…It is thought that E2 mediates the maintenance by it amino terminus-binding proteins that can bind host chromosomal DNA or, possibly, mitotic spindles (49,50). It is not known how HPV might bind mitotic spindles, but both BPV1 E2 and HPV16 E2 do bind the cellular protein ChlR1, a helicase that itself binds the cohesin complex (51,52). This binding, however, appears not to mediate chromosomal tethering (51).…”