Little is known about the immediate early events of the human papillomavirus (HPV) lifecycle. This is mostly due to the lack of a cell culture model allowing efficient infection of primary keratinocytes and at the same time a genetic analysis of viral factors. We have recently described such a system using 293TT-derived HPV16 quasivirions and extracellular matrix (ECM)-to-cell transfer that achieves highly efficient infection of primary keratinocytes. Infected cells support the complete viral lifecycle when subjected to growth in organotypic raft cultures and the system is amenable to genetic screening of viral factors. Current models suggest that the human papillomavirus undergoes three modes of replication during a complete life cycle. During the establishment phase, incoming viral genome is amplified to reach up to several hundred episomal genome copies. During maintenance, genome copy number is maintained. Mechanistically, post-transcriptional and -translational regulation of the viral E1 replication function as well as the E8^E2 repressor have been suggested to be important for genome maintenance. Lastly, differentiation-induced genome amplification in the late stages of the viral life cycle results in amplification of viral genome to thousands of copies. Rather than using a bidirectional mode of replication, a rolling circle mode of replication was suggested. Using an infection model combined with highly sensitive in situ detection of viral genome, which we developed in the last funding period, we have recently made unexpected discoveries: (i) HPV16 and HPV31 viral genome is amplified in each S phase during the maintenance stage in monolayer cell culture; (ii) a significant fraction of viral genome is lost to the cytosol during mitosis by failing to tether to mitotic chromosomes; lost nuclear viral genome is replenished in the next S phase; (iii) cytosolic viral genome is also detected in organotypic raft cultures; (iv) cytosolic viral genome is degraded during G1 and S phase by a lysosomal pathway likely utilizing (micro)autophagy; (v) E7 protein is required for efficient degradation of the cytosolic viral genome; (vi) cytosolic viral genome fails to induce cGAS/STING signaling despite a functioning cGAS/STING signaling pathway in HPV harboring keratinocytes. Our findings suggest that genome maintenance is regulated at the tethering level. Based on our findings, we hypothesize that genome copy number maintenance is regulated through restricted genome tethering to host cell chromosomes during mitosis rather than being regulated by replication initiation. The degradation of cytosolic genomes resets genome copy number after each mitosis. This immediately opens questions that regard the factor(s) that are limiting viral genomes tethering to mitotic chromosomes, the mechanism by which degradation of cytosolic viral genome is occurring, and why cytosolic viral genome does not induce innate immune signaling. We propose three specific aims to test...