Date Published: August 31, 2016
Publisher: Public Library of Science
Author(s): Irena Zurnic, Sylvia Hütter, Ute Rzeha, Nicole Stanke, Juliane Reh, Erik Müllers, Martin V. Hamann, Tobias Kern, Gesche K. Gerresheim, Fabian Lindel, Erik Serrao, Paul Lesbats, Alan N. Engelman, Peter Cherepanov, Dirk Lindemann, Michael Emerman.
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.
As obligatory intracellular parasites, retroviruses depend on host cell machineries for successful replication. Over the years, many cellular proteins counteracting or aiding retroviral replication have been identified [reviewed in 1, 2]. Expectedly, a large number of these factors are capsid protein interaction partners [reviewed in 3, 4, 5]. Since retroviral Gag proteins orchestrate numerous intracellular trafficking steps and viral budding, they naturally recruit numerous cellular factors and machineries to accomplish their functions.
The timing of integration of some retroviruses including MLV and FVs has long been recognized to coincide with mitosis [reviewed in 51], but until now no explanation on how this process is coordinated has been offered. Since host cell division is a major limiting factor for the broad use of FV (and some other retroviral) vectors for transduction of resting target tissues, knowledge of the cause of this limitation may help to improve the existing gene therapy vector systems .