Proceedings of the Frontiers of Retrovirology Conference 2016

Abstract

Question: Foamy viruses (FV), and in particular PFV, have emerged in recent years as attractive gene therapy vector candidates. Since the lack of knowledge on molecular events in FV replication is a major hurdle for broader usage of foamy virus vectors, we aimed at elucidating PFV biology by investigating interactions of its capsid protein, Gag, with host cell components. Methodology and result: To this end, we identified members of the mammalian PLK family as PFV Gag interactants in a commercial yeasttwo-hybrid (Y2H) screen and validated these results in detailed Y2H experiments for PLK1-3. In the yeast system, the intact PLK kinase and substrate recognition motifs were required for interactions with PFV Gag, in which a unique S 224 -T-P 226 motif served as a PLK binding determinant. PFV Gag mutants harbouring alanine substitutions of STP residues (iSTP) or phosphomimetic mutations of the T 225 (pmSTP) failed to interact with PLK1-3 in yeast. These findings were corroborated by colocalization studies of ectopically expressed, fluorescently tagged proteins in mammalian cells, where mCherry-tagged PFV Gag was able to recruit eGFP-tagged PLK1 and 2 to condensed mitotic chromatin in an STP motif-dependent manner. When characterizing PFV virions containing wild type or STP mutant Gag proteins, we observed that the mutations did not interfere with particle assembly, release or reverse transcription, but led to a 70 % titer reduction relative to wild type in single-round infection experiments. These replication defects became more prominent in the replication-competent PFV context. Therefore, the lack of Gag STP mutant interaction with PLK proteins upon viral entry into host cells was likely underlying this replication deficit. This hypothesis was strengthened by the finding that enzymatic PLK inhibition in host cells during transduction with wild type PFV mimicked the replication phenotype of PFV STP mutants. In addition to the overall reduced infectivity of the mutants, we also observed that the STP mutations in particle-associated Gag lead to differential sensitivity to integrase inhibition by dolutegravir and resulted in decreased integration efficiency. Conclusions: Taken together, our results demonstrate that PLK proteins influence PFV replication by virtue of their interaction with the Gag protein, ensuring timely and efficient transduction.