Single-cell analysis identifies TOP2α as a critical regulator of G2/M entry and differentiation-dependent productive HPV replication

High-risk human papillomaviruses (HPVs) are the etiological agents of over 5% of cancers worldwide including those of the cervix and oropharynx. HPVs infect stratified epithelia and establish latent infections in basal cells but restrict productive replication or amplification to differentiated suprabasal cells. Despite the presence of viral genomes in most differentiated cells, only a subset of cells amplifies genomes as well as express late genes and these correspond to those that have re-entered G2/M, but the factors regulating this selectivity are unknown. To determine the signals that control the productive viral life cycle, single cell RNA seq was performed on cells that stably maintain high-risk HPV genomes following differentiation. Ten populations of undifferentiated and differentiating keratinocytes were identified, however, only one differentiated population had entered G2/M, expressed late genes and amplified viral genomes. One of the highly expressed replication factors in this population was the type II topoisomerase TOP2 while no other topoisomerases were similarly induced. TOP2 was found to bind to viral genomes, and acute depletion in differentiating cells blocked entry into G2/M, impaired genome amplification and abrogated late gene expression. Amplification also requires activation of DNA repair pathways through the induction of high levels of DNA breaks, and TOP2 accounted for more than half of the breaks present in differentiating cells. Increases in levels of TOP2 in differentiating cells were driven by the E7 oncoprotein acting through the transcription factor FOXM1, which also controls expression of G2/M factors suggesting an auto-regulatory loop. These studies identify TOP2 as a critical regulator of HPV genome amplification upon differentiation.