E8^E2 controls the formation of E2-positive replication centers that precede E4 expression during productive HPV16 infection

Human papillomaviruses (HPVs) couple their replication cycle to keratinocyte differentiation. Viral genomes are maintained at low copy numbers in undifferentiated cells and undergo amplification within nuclear replication foci in differentiated suprabasal cells. The viral proteins E2 and E8^E2 function as activators and repressors of viral replication, respectively. Using a novel polyclonal antiserum together with the monoclonal antibody 22E2-B9, which recognize both E2 and E8^E2, we identified distinct nuclear E2 foci in keratinocytes harboring episomal HPV16 wild-type (wt) or E8^E2 mutant genomes. These foci colocalized with viral genomes and the cellular proteins RPA32, RAD51, γH2AX, and BRD4, consistent with viral replication centers. Cell-cycle analysis showed that E2 foci formed during the S phase and persisted into the G2 phase. Loss of E8^E2 expression or repression activity increased the number of replication foci and induced diffuse nuclear E2 staining. Only a subset of E2 focus-positive cells expressed the late viral protein E4, indicating that replication centers arise before robust late gene expression. In organotypic cultures, E2 focus-positive/E4-negative cells were detected in suprabasal layers before E2 focus-positive/E4-positive cells, whereas E2-negative/E4-positive cells suggested dynamic regulation of E2 during productive infection. Moreover, many wt cells displayed E2 focus phenotypes resembling E8^E2 mutant cells, suggesting that E8^E2 activity declines during keratinocyte differentiation. These findings demonstrate that E2-positive viral replication centers are tightly regulated by E8^E2, emerge early during productive HPV16 infection, and are accompanied by dynamic modulation of E2 and E8^E2 during the productive phase of the viral life cycle. IMPORTANCE: Human papillomaviruses (HPVs) couple their replication cycle to keratinocyte differentiation, but the mechanisms controlling productive viral genome amplification remain incompletely understood. Here, we identify distinct E2-positive nuclear replication centers in HPV16-infected keratinocytes and show that they contain viral genomes and cellular factors involved in DNA replication and repair. We demonstrate that these structures form during the S phase, persist into the G2 phase, and arise before robust expression of the late viral E4 protein, indicating that viral genome amplification is initiated earlier in the productive cycle than previously appreciated. We further show that the viral repressor E8^E2 regulates the organization of E2-positive replication centers and that E8^E2 activity appears to decline during keratinocyte differentiation. Together, our findings reveal dynamic regulation of E2 and E8^E2 during productive HPV16 infection and provide new insights into how viral genome amplification and late gene expression are coordinated in differentiating epithelial cells.