Polyomaviruses can either infect cells, replicate, and induce cell death upon lysis (permissive cells) or infect cells and transform them rather than undergoing viral replication (non-permissive cells). In permissive cells, the virion attaches to specific surface receptors, enters the cell and is transported to the nucleus, where viral DNA is released. Early phase proteins such as large T antigen drive the cell into S phase, then use cellular DNA synthesis proteins such as DNA polymerase and thymidine kinase to replicate. T antigen binding then initiates transcription of late viral RNAs for proteins such as VP1, VP2, and VP3, and progeny virions are assembled in the nucleus. Host cell lysis then releases the viral particles.

In non-permissive cells or permissive cells infected with defective viral genomes, the tumor antigens are synthesized, and cellular DNA synthesis is stimulated, but late viral capsid genes are not expressed. Instead, the viral genome integrates randomly into the host chromosome. In SV40, the large T antigen binds p53 and RB proteins to trigger immortalization. In other polyomaviruses, the middle T antigen binds cellular proteins such as c-src, and promotes cellular replication.