Mus musculus papillomavirus 1 E8∧E2 represses expression of late protein E4 in basal-like keratinocytes via NCoR/SMRT-HDAC3 co-repressor complexes to enable wart formation in vivo

High-risk human papillomaviruses (PV) account for approximately 600,000 new cancers per year. The early protein E8 boolean AND E2 is a conserved repressor of PV replication, whereas E4 is a late protein that arrests cells in G2 and collapses keratin filaments to facilitate virion release. While inactivation of the Mus musculus PV1 (MmuPV1) E8 start codon (E8-) increases viral gene expression, surprisingly, it prevents wart formation in FoxN1(nu/nu) mice. To understand this surprising phenotype, the impact of additional E8 boolean AND E2 mutations was characterized in tissue culture and mice. MmuPV1 and HPV E8 boolean AND E2 similarly interact with cellular NCoR/SMRT-HDAC3 co-repressor complexes. Disruption of the splice donor sequence used to generate the E8 boolean AND E2 transcript or E8 boolean AND E2 mutants (mt) with impaired binding to NCoR/SMRT-HDAC3 activates MmuPV1 transcription in murine keratinocytes. These MmuPV1 E8 boolean AND E2 mt genomes also fail to induce warts in mice. The phenotype of E8 boolean AND E2 mt genomes in undifferentiated cells resembles productive PV replication in differentiated keratinocytes. Consistent with this, E8 boolean AND E2 mt genomes induced aberrant E4 expression in undifferentiated keratinocytes. In line with observations for HPV, MmuPV1 E4-positive cells displayed a shift to the G2 phase of the cell cycle. In summary, we propose that in order to enable both expansion of infected cells and wart formation in vivo, MmuPV1 E8 boolean AND E2 inhibits E4 protein expression in the basal keratinocytes that would otherwise undergo E4-mediated cell cycle arrest. IMPORTANCE Human papillomaviruses (PVs) initiate productive replication, which is characterized by genome amplification and expression of E4 protein strictly within suprabasal, differentiated keratinocytes. Mus musculus PV1 mutants that disrupt splicing of the E8 boolean AND E2 transcript or abolish the interaction of E8 boolean AND E2 with cellular NCoR/SMRT-HDAC3 co-repressor complexes display increased gene expression in tissue culture but are unable to form warts in vivo. This confirms that the repressor activity of E8 boolean AND E2 is required for tumor formation and genetically defines a conserved E8 interaction domain. E8 boolean AND E2 prevents expression of E4 protein in basal-like, undifferentiated keratinocytes and thereby their arrest in G2 phase. Since binding of E8 boolean AND E2 to NCoR/SMRT-HDAC3 co-repressor is required to enable expansion of infected cells in the basal layer and wart formation in vivo, this interaction represents a novel, conserved, and potentially druggable target.