The papillomavirus (PV) life cycle is strictly controlled and can be divided into the following three distinct stages: initial infection, maintenance, and amplification. The papillomavirus E2 gene encodes a multifunctional protein responsible for regulating transcription and replication by recruiting viral and host factors to the viral DNA genome. Our lab has previously reported that tyrosine 102 may impact bovine (BPV) and human (HPV) viral replication in cell culture systems. This tyrosine is conserved in the E2 protein of the murine papillomavirus MmuPV1. To investigate how this amino acid impacts the MmuPV1 lifecycle in vivo, we generated potential phosphorylation mimetic (Y102E) and phosphorylation deficient (Y102F) mutants in the E2 open reading frame. The Y102F mutant protein supported both transcriptional activation and transient replication, while Y102E was defective. However, Y102E was capable of associating with E1 and the Brd4 C-terminal motif. When these E2-mutated MmuPV1 genomes were introduced into the skin of immunocompromised mice, only Y102F was capable of inducing papilloma development and production of infectious progeny virus. These findings demonstrate that while highly conserved, tyrosine at this position is not required by the virus. These data suggest that the chemical nature of the amino acid at this position can influence E2 activity and viral replication.