Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration
[['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 .', 'Similar to its orthoretroviral relatives , prototype foamy virus ( PFV ) , the best-studied member of the genus spumavirus in the Spumaretrovirinae subfamily , relies on Gag interactions with host cell proteins for successful replication [reviewed in 6] .', 'However , 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] .', 'To our knowledge , only six host factors that interact either directly or indirectly with PFV Gag have been identified , but given the multitude of roles PFV Gag accomplishes for viral replication in cells , this list is clearly far from complete .', 'The tumor susceptibility gene 101 ( Tsg101 ) component of the ESCRT-I complex directly interacts with primate FV Gag proteins to promote efficient virus budding [9 , 10] .', 'Furthermore , direct or indirect interactions with actin and core histones ( H2A and H2B ) were identified upon co-precipitation of these cellular proteins with ectopically expressed PFV Gag [11 , 12] .', 'These interactions are thought to be involved in PFV Gag precursor protein processing regulation and facilitate docking of the PFV pre-integration complex ( PIC ) to host cell chromosomes , respectively .', 'FVs are also known to be restricted in a species-specific manner by cellular Trim5α proteins [13–15] .', 'The viral determinants essential for restriction were mapped to the N-terminus of FV Gag , which probably engages Trim5α in the context of the viral capsid .', 'In addition , a member of the DEAD-box helicase family , DDX6 , was found to co-localize with PFV Gag at assembly sites and aid in viral RNA encapsidation into newly assembling FV particles [16] .', 'Finally , a potentially direct interaction of dynein light chain 8 with a coiled-coil domain within the N-terminus PFV Gag was reported , which appears to facilitate transport of incoming capsids to the centrosome of host cells [17] .', 'At the centrosome of non-dividing cells PFV capsids can remain intact and biologically active for several weeks [18] .', 'PFV capsids seem to proceed only to further disassembly steps upon entry of the cell into mitosis , which is required for PFV genome integration into cellular chromatin .', 'The cues and machineries coordinating viral uptake and intracellular trafficking are incompletely characterized .', 'Until now , it has only been speculated that members of cell cycle regulatory pathways may aid these processes .', 'One family of mitosis promoting proteins is the polo-like kinases ( PLKs ) , which are direct downstream effectors of cyclin/cyclin-dependent kinase complex ( Cyclin/CDK ) cascades .', 'PLK proteins are cellular Ser/Thr protein kinases that in vertebrates comprise five paralogues , PLK1-5 [reviewed in 19 , 20 , 21] .', 'They are crucial regulators of cell cycle progression , centriole duplication , mitosis , cytokinesis and DNA damage response .', 'However , other functions unrelated to cell cycle control such as protein aggregation and cellular stress response have also been attributed to various PLKs [22 , 23] .', 'Mammalian PLK proteins are characterized by a modular structure ( Fig 1A ) , comprising an N-terminal ( Ser/Thr ) catalytic kinase domain ( KD ) and a C-terminal polo-box domain ( PBD ) .', 'The PBD , which harbors one or more polo-boxes ( PBs ) that coordinate substrate binding , regulates catalytic activities and subcellular localization of PLKs [24–26] .', 'With the exception of PLK4 , which contains a unique PBD organization [27] , the PLK PBDs facilitate binding to phosphopeptides with an optimal Ser- ( pThr/pSer ) -Pro ( S- ( pT/pS ) -P ) substrate binding motif [28 , 29] .', 'The best-studied member of the PLK family is PLK1 , which typically utilizes its PBD to target proteins that have been primed through phosphorylation at their PLK binding site by other cellular kinases , such as CDKs or MAPKs [29 , 30] .', 'However , there are a few cases known where the PLK1 PBD binds non-phosphorylated peptides or phosphopeptides that were previously phosphorylated by PLK1 itself [30 , 31] .', 'Thus , multiple mechanisms exist to modulate the function and interaction of PLK1 and other PLKs and their targets in space and time .', 'Although replication of various viruses is influenced by cellular PLKs and viruses have been reported to modify the activity of PLKs for their replication , only a few direct interactions of viral proteins with PLKs have been reported .', 'For example the P protein component of the viral RNA dependent RNA polymerase ( vRdRp ) of paramyxoviruses , such as parainfluenza virus 5 ( PIV5 ) or mumps virus ( MuV ) , harbors a PLK1 STP binding site and is phosphorylated at another site by PLK1 [32 , 33] .', 'PLK1-mediated P protein phosphorylation downregulates viral gene expression and thereby avoids efficient induction of the host innate immune response [34] .', 'Furthermore , the interaction of the E2 protein of human papillomavirus type 5 with PLK1 was reported to inhibit Brd4 phosphorylation by PLK1 , thereby interfering with cellular functions of Brd4 in promoting cell cycle progression from the G1 to the S phases [35] .', 'Finally , binding of PLK1 to the NS5A protein of hepatitis C virus and its hyperphosphorylation by PLK1 is important for efficient virus replication [36] .', 'To gain further insight into the cellular biology of PFV replication and to identify new potential novel Gag interactors , we conducted a yeast two-hybrid ( Y2H ) screen using Gag protein as bait against different human cDNA expression libraries .', 'In addition to the known PFV Gag interactor Tsg101 , a member of the PLK family was identified as a new hit .', 'A detailed characterization and analysis revealed that PFV Gag specifically interacts with cellular PLK1 and PLK2 and alters their intracellular distribution .', 'PFV particles , unable to interact with cellular PLKs upon virus entry , display an infectivity defect manifested by a delayed and reduced capacity to integrate viral genomic DNA ( vgDNA ) into host cell chromatin .'], ['To elucidate intracellular steps of PFV replication in more detail , we performed a Y2H screen , aiming to identify novel Gag protein interaction partners .', 'Full-length ( FL ) PFV Gag ( PG ) was used as bait , fused either to the N- or C-terminus of the GAL4 DNA binding domain ( DB , here referred to as pNGB PG/coPG and pCGB PG/coPG , respectively ) ( Fig 1B ) .', 'These bait constructs were used and tested for interaction with a normalized HeLa cDNA expression library and two different full-length human open reading frame ( ORF ) expression libraries , where prey proteins were fused to the C-terminus of the GAL4 activation domain ( AD , pGADT7 , here referred to as pCGA ) .', 'Several human proteins were identified as likely PFV Gag interactors .', 'Among them was the known PFV Gag interactor , Tsg101 ( 7 different fragments , identified a total of 51 times ) , previously shown to interact with PFV Gag in Y2H experiments [10] .', 'A potential new PFV Gag interactor identified multiple times in the screen was human PLK2 ( hPLK2; 7 different fragments spanning the C-terminal PBD , identified a total of 20 times ) .', 'We note that the hPLK2 PBD scored in the assay only in combination with the C-terminally tagged FL PFV Gag protein bait ( pNGB PG/coPG ) , whereas Tsg101 was identified with both PFV Gag baits .', 'Additional Y2H experiments were performed to validate the results , investigate whether PFV can interact with other members of the PLK family , and characterize essential interaction domains/motifs within both proteins .', 'The analyses included various bait constructs encoding FL PFV Gag as well as point mutants or N- and C-terminal truncations , fused to the N- or C-terminus of the GAL4 DB ( Fig 1B ) .', 'Prey constructs encoded FL ORFs of hPLK1-3 , 5 , mouse PLK5 ( mPLK5 ) , rat PLK2 ( rPLK2 ) and point mutants thereof , as well as the PBDs of hPLK1-4 , fused to the C-terminus of the GAL4 AD ( pCGA-PLKs ) ( Fig 1A and 1B ) .', 'A prey encoding the FL Tsg101 ORF was utilized as positive control , whereas negative controls included the empty bait ( pNGB , pCGB ) and prey ( pCGA , pNGA ) vectors .', 'The results of the Y2H analysis are summarized in Fig 2 and S1 Fig FL PFV Gag interacted with FL hPLK2 and 3 , rPLK2 as well as PBDs of hPLK1-3 ( Fig 2A and 2B ) .', 'FL PFV Gag did not interact with FL hPLK1 , though PFV Gag baits with C-terminal deletions of 104 or more residues did interact with hPLK1 ( Fig 2A; S1A and S1B Fig ) .', 'Interactions with other tested PLK proteins were not detected ( Fig 2; S1 Fig ) .', 'These results indicate that FL PFV Gag can interact with various hPLKs and that these interactions are mediated via the kinase PBDs .', 'The use of N- and C-terminal Gag bait truncations allowed characterization of a minimal essential interaction domain spanning amino acids 130–295 ( Fig 2A; S1 Fig ) .', 'A closer inspection of this region revealed the presence of a Ser-Thr-Pro ( S-T-P ) motif at position 224–226 , which was unique within the FL PFV Gag protein ( Fig 1C ) .', 'Interestingly , this motif , which matches the minimal consensus PLK recognition and binding site ( S-T/S-P ) [29] , is present in all primate FV Gag proteins ( Fig 1C ) .', 'Exchange of any of the residues of the PFV Gag STP motif in context of the FL protein , including inactivating ( iSTP ) and phosphomimetic ( pmSTP ) mutations at T225 , abolished interactions with all PLK preys that interacted with the wild type ( wt ) Gag protein , without affecting the interaction with Tsg101 ( Fig 2A and 2B ) .', 'Hence , a preserved PFV Gag STP motif seems to be essential for interaction with cellular PLK proteins .', 'In order to determine which domains and enzymatic properties of the PLK proteins are important for the interactions with PFV Gag , we focused on FL rPLK2 ORFs with point mutations in critical residues as preys ( Fig 2B; S1C Fig ) .', 'As expected , none of the iSTP or pmSTP Gag constructs interacted with any of these rPLK2 variants , confirming interaction dependency on the PFV Gag STP motif .', 'FL wt Gag protein interacted with wt and constitutively active T236E ( caKD ) rPLK2 , whereas no interaction was detected with kinase inactive K108M ( iKD ) rPLK2 or its PBD mutant W504F ( iPBD ) ( Fig 2B ) .', 'These results suggest that the kinase activity of rPLK2 is required for stable interaction with FL PFV Gag , which is mediated by the rPLK2 PBD .', 'Examination of the amino acid sequence of the other PFV structural proteins revealed the presence of two additional consensus STP peptide motifs in Pol but none in the cytoplasmic domains of Env .', 'Both putative Pol PLK binding sites are located in the integrase domain of Pol surrounding T961 and T1058 ( S2A Fig ) .', 'However , an extensive Y2H interaction analysis using bait constructs expressing either a PFV Pol precursor protein with enzymatically inactive PR or the mature integrase subunit failed to demonstrate signs of interaction with any of the PLK prey constructs that showed interactions with PFV Gag ( S2B Fig ) .', 'Principal protein interaction functionality of the employed Pol and integrase bait constructs was demonstrated through their positive interaction , probably as a consequence of Pol/integrase oligomerization when using respective bait and prey construct combinations ( S2B Fig ) .', 'To confirm the interaction between Gag and PLK1-3 in human cells , we investigated localization patterns of ectopically expressed , fluorescently labeled proteins .', 'Constructs encoding C-terminally mCherry-tagged FL PFV Gag proteins ( either wt or iSTP/pmSTP variants ) and N-terminally eGFP-tagged variants of either FL hPLK1-3 or rPLK2 proteins were co-transfected into 293T cells .', 'Representative results of confocal fluorescence microscopy analysis for selected Gag-PLK combinations are shown in Fig 3 and S3 Fig . Introduction of single mutations into the PFV Gag STP motif had no effect on intracellular Gag localization .', 'As described previously [37] , all PFV Gag variants bound to chromatin during mitosis , completely overlapping the DAPI chromatin signal ( Fig 3B and 3C ) .', 'In line with previous observations , eGFP-tagged PLKs displayed speckled nuclear and diffuse cytoplasmic distributions when expressed on their own ( Fig 3A and 3D; S3A Fig ) [38] .', 'Importantly , none of the PLKs showed the overall chromatin association that was observed for PFV Gag ( Fig 3A and 3C; S3A Fig ) .', 'Strikingly , the subcellular localization of hPLK1 , 2 and rPLK2 , and to less of an extent also hPLK3 , was dramatically altered during mitosis in the presence of wt but not iSTP PFV Gag ( Fig 3B; S3B and S3C Fig ) .', 'Wt PFV Gag recruited PLK1 and PLK2 proteins to mitotic condensed chromatin resulting in an almost complete overlap of both signals , whereas for hPLK3 only a weak co-localization was observed .', 'Distinct co-localization was not observed between any of the Gag variants with the examined eGFP-PLK fusions in interphase cells ( Fig 3D and 3E; S3 Fig ) .', 'In some cases , 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 ) .', 'Occasionally , co-localization between eGFP-hPLK1 and Gag-mCherry variants was noted at a perinuclear region , presumably the centrosome ( Fig 3E , white arrow ) .', 'However , 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 ) .', 'As these results established that an intact STP motif in PFV Gag was required for co-localization with hPLK1 and hPLK2 in human cells , we addressed next whether the kinase requirements would correspond to the Y2H data using rPLK2 as a representative candidate .', 'Hence , the same set of rPLK2 variants involved in the Y2H interaction analysis was employed in combination with wt and T225A PFV Gag .', 'Whereas wt Gag , but not its T225A variant , co-localized with wt ( wt ) and constitutively active rPLK2 ( caKD ) , co-localization was not observed for any other rPLK2 protein variant including the kinase inactive ( iKD ) mutant ( Fig 3B; S3D Fig ) .', 'Taken together , our results confirm that PLK1 and 2 and to a limited extent also PLK3 are cellular interaction partners of PFV Gag .', 'The viral structural protein appears to recruit the PLKs to condensed , mitotic chromatin in an STP motif-dependent fashion , which requires functional PLK KD and PBDs .', 'The interaction of PLKs with substrates is reportedly strongly dependent on PLK binding site phosphorylation status [29] .', 'Nonetheless , low affinity binding to non-phosphorylated binding sites , and a few binding sites phosphorylated by PLKs themselves , have been reported [reviewed in 39] .', 'To determine the phosphorylation-status of the putative PFV Gag PLK STP binding site , we examined particle-associated Gag or protein immunoprecipitated from cell lysates with various phosphopeptide-specific antibodies , including a custom-made antiserum specific for the PFV Gag STP motif ( Fig 4 ) .', 'Detectable amounts of phosphorylated Gag protein were absent in immunoprecipitates of lysates of 293T cells transiently transfected with expression constructs of the four-component PFV vector system .', 'This suggests that the majority of cellular PFV Gag is not phosphorylated at threonine residues in the context of T-P motifs , including the PLK binding motif .', 'In contrast , phosphorylated Gag was readily detected in lysates of released wt PFV virions pelleted by ultracentrifugation , by either a commercial pThr-Pro ( pT-P ) –specific monoclonal or the custom-made PFV Gag PLK binding site phospho-specific ( Gag S-pT-P ) polyclonal antibody ( Fig 4B–4D ) .', 'pT-P-specific signal intensity was reduced in particle lysate samples of all iSTP/pmSTP mutants , except S224A , which theoretically can still be phosphorylated at T225 by a non-PLK cellular priming kinase ( Fig 4B ) .', 'Furthermore , pT-P-specific signals were almost undetectable in both wt and T225A mutant particle lysates pretreated with phosphatase ( Fig 4C ) .', 'Hence , T225 phosphorylation appears to contribute to a large extent to the PFV Gag pT-P-specific signal detected in wt PFV particle samples .', 'This was further substantiated by results using the custom-made PFV Gag S-pT-P-specific antiserum .', 'A strong signal was detected in wt PFV particle samples , which was largely reduced by phosphatase pretreatment down to the background levels that were observed in PFV Gag T225A particle samples regardless of phosphatase treatment ( Fig 4D ) .', 'In concordance with the results of Western blotting , mass spectrometric analysis revealed that a fraction of the PFV Gag protein recovered from SDS-PAGE-separated wt PFV particle samples is phosphorylated at the 223-TST-225 cluster ( S4A and S4B Fig ) .', 'Obtained high-resolution fragmentation spectra do not exclude that within the 223–225 cluster the phosphogroup is localized at the position 225 ( S4C Fig ) .', 'Taken together , these results indicate the presence of significant amounts of PFV Gag protein with phosphorylated PLK binding site motif in released wt PFV virions , which can potentially serve as a high affinity interaction partner for cellular PLK proteins upon PFV entry into newly infected host cells .', 'The interaction between PFV Gag and human PLK proteins has not been implicated in previous studies , but may be of importance for the cell cycle-dependent replication characteristics of PFV [40–42] .', 'Hence , we investigated whether introduction of Gag iSTP/pmSTP variants into PFV particles may exert an effect on virus replication .', 'The infectivity of the PFV Gag mutants was assessed in the context of both replication-competent and single-round PFV expression systems , with similar results obtained between systems .', 'Inclusion of any of the tested STP Gag mutations into the packaging construct decreased infectivity of respective single-round PFV vector supernatants on HT1080 , human primary fibroblast MRC-5 cells or murine embryonic fibroblasts by 50–70% ( Fig 5A; S5A Fig ) .', 'A similar infectivity defect was observed early after target cell infection for replication-competent mutant viral particle preparations generated from full-length proviral expression constructs ( Fig 5B ) .', 'Interestingly , the replication defect of the Gag STP motif mutant viruses became more prominent upon prolonged growth of the infected cell cultures ( Fig 5C ) .', 'The infectivity of iSTP/pmSTP Gag-containing particles progressively mitigated down to 2 to 8% relative to wt after 10 days of co-cultivation .', 'A similar effect was also observed in human primary fibroblast MRC-5 cells ( S5B Fig ) , indicating the phenotype is not cell-type specific .', 'Thus , PFV particles harboring Gag unable to interact with cellular PLKs display a diminished infectivity in single-round infections and fail to efficiently spread in target cell cultures of different origin .', 'Even though we were unable to detect any signs of PFV Pol interaction with cellular PLKs in the Y2H analysis , we nevertheless examined the influence of various PFV Pol STP motif mutants on viral infectivity in combination with either wt or iSTP mutant PFV Gag ( S2C Fig ) .', 'Unlike Gag iSTP mutant PFV particles , the Pol iSTP mutant particles neither showed a significant difference in viral infectivity nor did they further promote the Gag iSTP infectivity defect ( S2C Fig ) .', 'Taken together with the Y2H interaction analysis , these results strongly suggest that PFV Pol STP motifs located in the integrase domain did not mediate interactions with cellular PLKs and are dispensable for PFV-mediated infection of target cells .', 'In order to understand the underlying causes for the replication defects of PFV particles with abolished Gag-PLK interaction , we characterized various features of the mutant viruses .', 'We first harvested lysates of virus-producing cells after transient transfection of either four-component PFV vector system or full-length proviral PFV constructs into 293T cells .', 'We also studied purified viral particles released into the supernatant to examine viral protein expression , particle release , viral genomic RNA ( vgRNA ) packaging and intra-particle reverse transcription ( Fig 6; S6 Fig ) .', 'None of the PFV Gag STP motif mutations affected the efficiency of virus-production , as implicated by analysis of viral protein and nucleic acid content .', 'Particle release efficiency , viral protein composition and processing of these mutants were comparable to wt , as were their vgRNA and vgDNA contents in the context of both viral production systems ( Fig 6; S6 Fig ) .', 'Therefore , it can be concluded that the introduction of STP motif mutations into PFV Gag , which prevents interaction with cellular PLK proteins , does not have an effect on virus production , release , vgRNA packaging or intra-particle reverse transcription activity .', 'The observed infectivity defects of PFV Gag STP motif mutant viruses pointed to an essential function of Gag-hPLK interactions at an early stage in viral replication .', 'The results suggest that phosphorylation of a viral and/or cellular target , after binding of cellular PLKs to the phosphorylated Gag S-pT-P binding site , may be beneficial for PFV infection .', 'To test this hypothesis we asked how enzymatic inhibition of hPLK1 might influence virus infectivity .', 'For inhibition of cellular PLKs , in particular PLK1 , during FV entry , HT1080 target cells were incubated with the well characterized PLK inhibitor BI-2536 [43] in two different regimens as outlined in Fig 7A .', 'Because BI-2536 treatment is known to arrest cells in prometaphase in a dose and time dependent manner , we used propidium-iodide ( PI ) staining to determine the cell cycle status 24 h post-infection , at the time when productive infection was quantified by flow cytometry ( Fig 7B ) .', 'Short-term treatment of HT1080 target cells with 50 nM BI-2536 for 5 h ( 1 h prior to and 4 h during virus incubation ) did not alter the cell cycle profile at 24 h post-infection as compared to the mock-treated population .', 'This indicates that 50 nM BI-2536 treatment yields a largely cycling cell population and that short-term hPLK1 inhibition did not result in cell cycle arrest .', 'In contrast , the target cell cycle profile was dramatically changed when 10 nM BI-2536 was applied for the entire 25 h examination period ( long-term treatment ) .', 'The target cell population was characterized by an altered cell cycle profile with highly increased G2-M population , indicating that hPLK1 inhibition by this inhibitor regimen led to a potent cell cycle arrest .', 'The influence of PLK inhibition by BI-2536 treatment revealed interesting , differential phenotypes for Gag wt and Gag STP mutant containing FVs ( Fig 7C ) .', 'When HT1080 target cells were treated with 50 nM inhibitor for only 5 h ( short-term ) the infectivity of wt PFV was reduced by 85% in comparison to mock treatment ( Fig 7C , orange bars ) .', 'In contrast , the infectivity of STP motif mutant particles ( T225A , T225E , P226A ) were only reduced by 25 to 30% in comparison to respective mock treated samples .', 'Thus , the short-term inhibitor treatment reduced the infectivity of wt virus nearly to the level of Gag STP mutant viruses .', 'A more prominent inhibitory effect was observed for the long-term 10 nM BI-2536 treatment regimen ( Fig 7C , magenta bars ) .', 'The infectivity of wt virus was again reduced to a much greater extent ( 65-fold ) than Gag STP mutant viruses ( 5–6 fold ) in comparison to the respective mock treatment controls , resulting in similar levels of residual infectivity .', 'Taken together the data demonstrate that short-term PLK1 inhibition during entry of wt PFV , which does not induce a cell cycle arrest , can largely mimic the infectivity defect of virus mutants with Gag proteins unable to interact with cellular PLKs .', 'The effect of BI-2536 on viral infectivity was also examined for murine leukemia virus ( MLV ) and HIV-1 ( HIV-1 ) vectors as representatives of retroviral genera that are dependent or independent of target cell mitosis for productive infection , respectively ( Fig 7C ) .', 'Both types of vectors were pseudotyped with PFV Env and harbored an identical reporter gene as transmitted by FV vector particles .', 'Whereas HIV-1 infectivity was only weakly ( 2-fold ) diminished under both treatment conditions , MLV infectivity was moderately inhibited ( 3-fold ) by short-term PLK inhibition and very strongly reduced ( 150-fold ) by long-term BI-2536 treatment .', 'Hence , the data indicate that hPLK1 inhibition effects are not PFV-specific , which may encourage further insight into PLK proteins as a key link between retroviral infection and cell cycle progression .', 'The reduced infectivity observed for PLK-interaction-deficient PFV or upon drug-mediated inhibition of PLKs during infection with wt PFV can result from defects at different steps during PFV entry and uptake into target cells .', 'This includes early steps such as attachment , membrane fusion , capsid stability or later steps such as stability of the PIC or its tethering to host cell chromatin as well as vgDNA integration .', 'To further narrow down which step might be affected we examined the uptake dynamics of wt and Gag T225A mutant virions containing fluorescently-tagged Gag proteins in synchronously infected HT1080 cells ( Fig 8 ) .', 'At different time points after initiating virus uptake the amount of cell-associated , fluorescently labeled viral Gag protein was quantified by flow cytometric determination of the mean fluorescent intensity ( MFI ) of the whole cell population .', 'As non-infectious control GFP-tagged Gag wt containing particles harboring a fusion-deficient PFV Env glycoprotein ( iFuse ) were used .', 'All types of virions similarly attached to target cells by spinoculation at low temperature ( Fig 8 ) .', 'Fusion-deficient virions ( wt iFuse ) were rapidly internalized upon raising the temperature and the Gag-GFP signal declined to background levels within 4 to 24 h depending on the amount of virus used for infection .', 'This most probably is the consequence of rapid degradation of non-fused particles by endocytic uptake and targeting to lysosomes [44 , 45] .', 'In contrast , Gag wt ( wt wt ) and Gag T225A ( T225A wt ) samples showed as early as 1 h post-infection significantly higher MFI signals than the samples of fusion-deficient virions ( wt iFuse ) , and this difference further increased over time .', 'In line with previous studies [44 , 45] , this indicates rapid release of capsids into the cytoplasm for virions with fusion-competent , wt glycoprotein .', 'Cytoplasmic PFV capsids are known to be quite stable and transported along microtubules to the MTOC of the host cell , where they await host cell entry into mitosis for progression towards virus genome integration into cellular chromatin [18] .', 'In the examined time period of up to 24 h post-infection , no significant differences in the Gag-GFP MFI profiles of wt and Gag T225A mutant particles were observed , independent of the amount of virus initially attached to HT1080 cells .', 'Taken together the analysis indicates similar attachment , fusion and uptake dynamics as well as Gag protein stability for wt and Gag T225A virions .', 'This suggests a defect at a step post-disassembly of PFV capsids as responsible for the observed infectivity phenotype of iSTP mutant PFV virions .', 'Closer examination of reporter gene expression in target cells transduced with wt or Gag STP motif mutant PFV particles over time revealed marked differences in the severity of STP mutant infectivity defect ( Fig 9A ) .', 'The largest differences in STP motif mutant vector titers , up to 10-fold reduced compared to wt , were observed if the number of reporter gene expressing target cells used for virus titer calculation were quantified 1 day post-transduction in single-round infection experiments ( Fig 9A ) .', 'Up to 3 days post-transduction this difference constantly declined until reaching a plateau of 3-fold reduced infectivity in comparison to wt .', 'This phenotype of a decrease in viral infectivity and the time-dependent quantitative differences may suggest a combination of delayed and reduced integration potential of iSTP mutant PFV particles in comparison to wt virus .', 'To gain support for this hypothesis , we extracted genomic DNA from HT1080 cells 10 days post-infection with different virus supernatants and determined integrated PFV genome copy numbers by quantitative Alu PCR ( Fig 9B ) .', 'The number of integrated vector genomes of cells infected with PFV Gag T225A containing virus supernatants was reduced to 45% of wt and correlated well with their reduced infectivity .', 'Thus , the reduced viral titer of PFV Gag T225A containing particles determined by the reporter gene transfer assay and flow cytometric analysis late after transduction is mainly the consequence of a reduced number of proviral integration events .', 'Attenuated integration would suffice to explain the defects in mutant virus titers , but not the delay in the number of viral DNA-derived reporter gene expressing cells observed for the STP mutants during the first three days after infection .', 'To further characterize the iSTP/pmSTP integration dynamics , we made use of a well-characterized HIV-1 integrase inhibitor , dolutegravir ( DTG ) [46] , previously shown to also be active against PFV integrase [47] .', 'The rationale behind this set of experiments was that if wt and iSTP mutant viruses integrated their genomes at different dynamic rates during the initial 72 h post-infection , then they should be differentially sensitive to DTG treatment over time .', 'Following this notion , we synchronously infected HT1080 cells with wt ( wt wt ) or T225A mutant ( T225A wt ) PFV and mock solvent ( DMSO ) or 2 μM DTG at different time points post-infection and maintained the drug until flow cytometry analysis was performed at day 10 post-infection ( Fig 9C ) .', 'As control , cells were infected with supernatants harboring viral particles containing wt PFV Gag in combination with Pol variants with either enzymatic inactive reverse transcriptase ( wt iRT ) or integrase ( wt iIN ) domains .', 'As expected , DTG addition to samples infected with the latter viruses ( wt iRT , wt iIN ) did not influence their infectivity in comparison to respective mock treated controls ( Fig 9D ) .', 'In contrast , the infectivity of both Gag wt ( wt wt ) and Gag T225A ( T225A wt ) containing viral particles was efficiently inhibited ( to ~1% of respective mock treated samples ) when DTG was added at the time of virus addition ( 0 h time point ) ( Fig 9D ) .', 'Strikingly , the infectivity of both types of viruses was differently affected when DTG was added at later time points .', 'Gag wt ( wt wt ) containing particles became resistant to DTG-mediated inhibition quickly and acquired 50% drug resistance at ~12 h post-infection whereas Gag T225A ( T225A wt ) containing particles reached this value much later at ~27 h post-infection .', 'These results indicate that approximately half of wt PFV proviruses establish within 12 hours post-infection and confirm that Gag STP mutations cause a significant delay in PFV integration .', 'In sharp contrast to orthoretroviruses such as HIV-1 and MLV that predominantly integrate within or in the vicinity of actively expressed genes , PFV disfavors integration into transcriptionally active chromatin and within transcription units [48–50
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['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 .']
['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 .']
['phosphorylation', 'protein interactions', 'viral transmission and infection', '293t cells', 'cell cycle and cell division', 'biological cultures', 'cell processes', 'microbiology', 'viral structure', 'research and analysis methods', 'proteins', 'cell lines', 'viral replication', 'virions', 'biochemistry', 'host cells', 'cell biology', 'post-translational modification', 'virology', 'biology and life sciences', 'capsids']