Many viruses use alternate mechanisms to initiate protein translation owing to their limited coding capacity. The ribosomal protein S25 (RPS25/eS25) is required for efficient non-canonical mechanisms of translation initiation, such as internal ribosomal entry site (IRES) initiation or ribosomal shunting, but eS25 is not required for efficient cap-dependent initiation. Thus, eS25 knockdown can be used to evaluate whether a virus relies on alternative mechanisms of initiation. Since earlier studies suggest that simian virus 40 (SV40) uses an IRES to translate a minor capsid protein VP3, which is translated from the same transcript as VP2, we sought to test if BK polyomavirus (BKPyV) also used an IRES by examining viral production with and without eS25. Instead, we found that BKPyV required eS25 for robust viral production prior to gene expression, suggesting that it affected an early step in the viral life cycle. These studies revealed a role for eS25 in cell cycle control. When eS25 was knocked down in primary kidney cells, it decreased the proportion of cycling cells, causing arrest at both G0/G1 and G2/M. These data suggest that the timing of BKPyV infection depends on the initial cell cycle state of the host cell.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.