Simian virus 40 (SV40) was discovered in 1960 as a contaminant in early polio vaccines. Its discovery coincided with an explosion of knowledge in the new field of molecular biology, and SV40 was quickly adopted as a model to study eukaryotic genome structure, expression, replication, and cell growth regulation in cultured cells. With a genome of only 5.2 kbp, SV40 relies heavily on host cell machinery to propagate, affording investigators a powerful tool to discover key host proteins that the virus manipulates. Indeed, a single multifunctional viral protein, the large tumor (T) antigen (Tag) (Figure 1A), is sufficient to orchestrate the replication of the viral mini-chromosome in infected monkey cells. The origin DNA binding domain of Tag binds specifically to the viral origin of DNA replication, and the C-terminal helicase domain of Tag unwinds parental DNA at SV40 replication forks. The development of a cell-free reaction containing purified Tag and primate cell extract enabled the identification of ten evolutionarily conserved host proteins that are necessary and sufficient, together with Tag, to replicate SV40 DNA in vitro.