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Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells., Author Summary Viruses are masters at exploiting host cell machineries for their replication. For human immunodeficiency virus type 1 (HIV-1), the best-studied representative of the Orthoretrovirinae subfamily from the genus lentiviruses, numerous important virus-host interactions have been described. In contrast, only a few cellular proteins are known to influence the replication of foamy viruses (FVs, also known as spumaviruses), an intriguing type of complex retrovirus of the Spumaretrovirinae subfamily that combines features of both retroviruses and hepadnaviruses in its replication strategy. Given the increasing interest in FVs as gene transfer tools and their unique status within the retrovirus family, this discrepancy urged the identification of novel host cell interaction partners of FV structural components. This study focused on prototype FV (PFV), the best-characterized member of FVs, and its capsid protein, Gag, as the central player of viral replication. Members of the mitosis-regulatory, polo-like kinase (PLK) family were identified as novel Gag binding partners. The Gag interaction with PLK1 (and possibly also PLK2) facilitated efficient PFV genome integration into host chromatin, ensuring successful replication and viral spread in infected target cell cultures. Collectively, our results elucidate the first link between cell cycle regulatory networks and the mitosis-dependent PFV integration process.