Abstract
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.
Highlights
As obligatory intracellular parasites, retroviruses depend on host cell machineries for successful replication
This study focused on prototype foamy viruses (FVs) (PFV), the best-characterized member of FVs, and its capsid protein, Gag, as the central player of viral replication
The Gag interaction with PLK1 facilitated efficient PFV genome integration into host chromatin, ensuring successful replication and viral spread in infected target cell cultures
Summary
Retroviruses depend on host cell machineries for successful replication. Unlike for orthoretroviruses such as human immunodeficiency virus type 1 (HIV-1), few PFV Gag interaction partners have been identified to date This is surprising, given the peculiarities of the PFV replication cycle and their significance to understanding retroviral evolution and vector development [reviewed in 7, 8]. Overlap of mCherry and eGFP signals was observed in a diffuse pattern throughout the cytoplasm of interphase cells co-transfected with wt Gag and hPLK1, hPLK2, or rPLK2 (Fig 3E; S3B Fig). Co-localization between eGFPhPLK1 and Gag-mCherry variants was noted at a perinuclear region, presumably the centrosome (Fig 3E, white arrow) This event could not be ascribed to a specific co-localization pattern, as it was independent of the functional Gag STP motif and both partners individually localized to the microtubule-organizing center (MTOC) (Fig 3D)
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