Histone Variant macroH2A1 Spatially and Functionally Organizes Human Papillomavirus Replication Foci in the Productive Stage of Infection

The life cycle of HPV depends on keratinocyte differentiation as the virus modulates and takes advantage of cellular pathways to replicate its genome and assemble viral particles in differentiated cells. Viral genomes are amplified in nuclear replication foci in differentiated keratinocytes, and DNA repair factors from the DNA damage response signaling pathway are recruited to replicate viral DNA. The HPV genome is associated with cellular histones at all stages of the infectious cycle, and here we show the histone variant macroH2A1 is bound to the HPV genome and enriched in viral replication foci in differentiated cells. MacroH2A1 isoforms play important roles in cellular transcriptional repression, double strand break repair, and replication stress. The viral E8^E2 protein also binds to the HPV genome and inhibits viral replication and gene expression by recruiting NCoR/SMRT complexes. We show here that E8^E2 and SMRT also localize within replication foci, though not through direct interaction with macroH2A1. Conversely, transcription complexes containing RNA polymerase II and Brd4 are located on the surface of the foci. Foci generated with an HPV16 E8^E2 mutant genome are not enriched for SMRT or macroH2A1 but contain transcriptional complexes throughout the foci. We demonstrate that macroH2A1 promotes viral late transcription and propose that it does so by spatially separating replication and transcription activities within replication foci. IMPORTANCE Human papillomaviruses are small DNA viruses that cause chronic infection of cutaneous and mucosal epithelium. In some cases, persistent infection with HPV can result in cancer, and 5% human cancers are the result of HPV infection. In differentiated cells, HPV amplifies viral DNA in nuclear replication factories and transcribes late mRNAs to produce capsid proteins. However, very little is known about the spatial organization of these activities in the nucleus. Here we show that repressive viral and cellular factors localize within the foci to supress viral transcription, while active transcription takes place on the surface. The cellular histone variant macroH2A1 is important for this spatial organization. ### Competing Interest Statement The authors have declared no competing interest.