The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag
Nuclear localization of Gag is a property shared by many retroviruses and retrotransposons. The importance of this stage for retroviral replication is still unknown, but studies on the Rous Sarcoma virus indicate that Gag might select the viral RNA genome for packaging in the nucleus. In the case of Foamy viruses, genome encapsidation is mediated by Gag C-terminal domain (CTD), which harbors three clusters of glycine and arginine residues named GR boxes (GRI-III). In this study we investigated how PFV Gag subnuclear distribution might be regulated.
We show that the isolated GRI and GRIII boxes act as nucleolar localization signals. In contrast, both the entire Gag CTD and the isolated GRII box, which contains the chromatin-binding motif, target the nucleolus exclusively upon mutation of the evolutionary conserved arginine residue at position 540 (R540), which is a key determinant of FV Gag chromatin tethering. We also provide evidence that Gag localizes in the nucleolus during FV replication and uncovered that the viral protein interacts with and is methylated by Protein Arginine Methyltransferase 1 (PRMT1) in a manner that depends on the R540 residue. Finally, we show that PRMT1 depletion by RNA interference induces the concentration of Gag C-terminus in nucleoli.
Altogether, our findings suggest that methylation by PRMT1 might finely tune the subnuclear distribution of Gag depending on the stage of the FV replication cycle. The role of this step for viral replication remains an open question.
KeywordsFoamy virus Gag Nuclear trafficking Nucleolus Chromatin-binding Post-translational modification Methylation PRMT
Foamy viruses (FVs), also known as spumaviruses, are complex retroviruses that belong to the Spumaretrovirinae subfamily of the Retroviridae. They are endemic among many animal species, particularly non-human primates (NHPs) (for a review ). The Prototype FV (PFV) was isolated from human-derived cell culture and later found to be a chimpanzee FV . It is currently well established that humans are not natural hosts but acquire infection as a consequence of zoonotic transmission of simian FVs (SFVs) through bites of captive or wild NHPs [3, 4]. FV infection is persistent and apparently benign  and human-to-human transmission has never been reported. Like all retroviruses, FVs reverse transcribe their RNA genome (gRNA), which encodes the typical gag, pol and env genes, and integrate the resulting cDNA into the host cell chromosomes. However, specificities in the replication strategy of FVs set them apart from orthoretroviruses. These include the fact that reverse transcription occurs during viral particle production [5, 6], and that Pol is expressed independently of Gag from a specific spliced transcript [7, 8]. The structural organization and maturation profile of FV Gag are also peculiar. FV Gag lacks the major homology region (MHR) and the Cys-His zinc-finger motifs that are hallmarks of orthoretroviral Gag proteins. Moreover, FV Gag is not processed into the matrix (MA), capsid (CA) and nucleocapsid (NC) mature products like its orthoretroviral counterparts, but rather undergoes a single cleavage event that removes a 4 kDa C-terminal peptide (, reviewed in ). This feature is shared by the Gag proteins of the Drosophila retrovirus Gypsy  and the Ty1 retrotransposon of S. cerevisiae . Recent studies showed that PFV Gag N-terminal domain (NTD, amino acids (aa) 1–180) is entirely unrelated to its orthoretroviral counterpart . They also confirmed that the NTD, which harbors the cytoplasmic targeting and retention signal (CTRS) and the self-dimerization domain, plays a role similar to orthoretroviral CA in viral capsid assembly . In contrast the central conserved region of PFV Gag (aa 300–477), which is involved in the formation of higher-order multimers, shares a conformation analogous to that of orthoretroviral CA, suggesting evolution from a common ancestral protein . In the absence of structural data, functional studies indicate that the C-terminal domain (CTD, aa 400–648) of FV Gag plays a role related to that of orthoretroviral NC in genome packaging [10, 15]. This domain is enriched in glycine and arginine residues that in primate FV Gag proteins are clustered in three regions named GR boxes (GRI-III) . GRI binds nucleic acids in vitro and was proposed to be responsible of the incorporation of both the gRNA and Pol into virions [16, 17, 18]. The GRII box shows the highest conservation throughout evolution and is involved in the accumulation of PFV Gag in the nucleus . The determinant for nuclear localization within GRII maps to a 13-aa chromatin binding sequence (CBS, aa 534–546) that recognizes the H2A/H2B core histones. This interaction tethers the pre-integration complex (PIC) to host cell chromatin prior to viral integration [19, 20, 21]. The role of GRIII in FV replication is enigmatic but likely related to that of GRI, since the two motifs can functionally complement each other . Although the GR boxes were initially viewed as independent entities playing both specific and redundant functions, a recent study rather indicates that the positively charged residues within the CTD, not the GR boxes individually, mediate gRNA packaging and Pol encapsidation .
During FV replication Gag displays different subcellular localizations, as a result of numerous interactions with the intracellular trafficking machinery, which likely match its multiple roles throughout the viral life cycle. Our previous studies showed that, as a component of the incoming PIC, Gag drives the traffic of viral particles towards the microtubule-organizing center (MTOC) where uncoating occurs [24, 25, 26]. Upon nuclear envelope breakdown, Gag associates with host cell chromosomes and critically contributes to the selection of the integration sites [19, 20, 21, 27]. At a later stage, newly synthesized Gag molecules have been shown to oligomerize in the nucleus and, next, reach the cytoplasm by engaging the CRM1 (Chromosomal Maintenance 1, also known as Exportin 1)-dependent pathway through a nuclear export signal (NES) , as reported for Rous Sarcoma Virus (RSV) (see below, ). Currently, how newly synthesized Gag crosses the intact nuclear membrane is unknown .
Evidence that Gag proteins shuttle between the nucleus and the cytoplasm has been reported for other retroviruses, including RSV, feline immunodeficiency virus (FIV), Human Immunodeficiency Virus (HIV), mouse mammary tumor virus (MMTV), Mazon-Pfizer monkey virus (MPMV) and murine leukemia virus (MLV) ( and references therein), and the Tf1 retrotransposon . Gag or the isolated NCs of MMTV, RSV, FIV, HIV and MLV have also been detected in the nucleolus ( and references therein), a distinct subnuclear compartment that forms around the gene clusters encoding rRNAs and represents the site of ribosomes biogenesis. Although in most instances the significance of Gag nucleocytoplasmic trafficking for virus replication is elusive, RSV Gag was shown to oligomerize in the nucleus, in an RNA- and NC-dependent manner . The observation that nuclear trafficking of RSV Gag is required for efficient genome encapsidation [32, 33] and that binding to a synthetic oligonucleotide mimicking the packaging signal favours the association between Gag and the nuclear export factor CRM1 in vitro , led to propose a model according to which RSV Gag selects the viral gRNA for packaging in the nucleus.
To deepen our understanding of the nuclear trafficking of PFV Gag, we studied the localization of the C-terminal GR boxes and established that the isolated GRI and GRIII boxes are nucleolar localization signals (NoLSs). We also found that Gag localizes at least temporarily in the nucleolus during PFV replication. Next, we investigated the mechanisms that regulate this process, and identified the evolutionary conserved arginine residue at position 540 (R540) within the GRII box as a critical factor determining whether Gag localizes in the nucleolus or is tethered to chromatin. We also established that PFV Gag interacts with and is modified by PRMT1 (Protein Arginine Methyltransferase 1) and PRMT5. Interestingly, we found that PFV Gag harboring the R540A substitution is unable to interact with PRMT1 and lost the asymmetric dimethylarginine (ADMA) mark, while retaining binding to and modification by PRMT5. Finally, we observed that siRNA-mediated depletion of PRMT1 leads to nucleolar accumulation of the C-terminus of PFV Gag fused to GFP. On the basis of these results, we hypothesize that PRMT1-mediated methylation, which requires the invariant R540 residue, could regulate the subnuclear distribution of PFV Gag antagonizing nucleolar accumulation in favor of chromosome binding.
The GRI and GRIII boxes of PFV Gag are nucleolar localization signals
FV Gag transits through the nucleolus during viral replication
Since we could not exclude that the interaction between Gag and Gag-TRAP-GFP occurs outside the nucleolus and that the Rev NoLS within the chimeric protein subsequently targets the complex to this subcellular site, we analyzed Gag localization in PFV-infected U937MG cells treated with leptomycine B (LMB), a specific inhibitor of CRM1-mediated nuclear export, and/or exposed to hypoxia, a setting that was previously shown to slow down protein trafficking . Under these conditions, Gag (green staining) co-localized with nucleolin in about 3–10% of PFV-infected cells (Fig. 2d). Altogether these results indicate that Gag transits through the nucleolus during PFV replication.
The evolutionary conserved R540 residue is critical for both nucleolar localization and chromosome tethering of PFV Gag
We next studied the influence of R540 on full-length PFV Gag localization. PFV Gag expressed as GFP-fusion protein in HeLa cells was predominantly diffused in the cytoplasm of interphasic cells, and was excluded from the nucleolus (Fig. 3c, left panel). Upon deletion of the CBM or mutation of R540 to A, GFP-Gag accumulated in the nucleolus in a fraction of transfected HeLa cells (about 7 and 10%, respectively) (Fig. 3c, left panel). These results contrasted with the finding that GFP-GRsR540A was detected in the nucleolus in all the transfected cells (Fig. 3a, left panel). We hypothesized that this discrepancy might result at least in part from the presence of N-terminal sequences within Gag favoring its accumulation in the cytoplasm and/or antagonizing its nuclear/nucleolar localization. To address this point, we mutated three well-characterized functional domains in GFP-Gag or GFP-GagR540A, namely the CTRS (mutation R50A) , the NES (mutation G110 V)  and the dimerization domain (Δdim, deletion of aa 130–160)  (Fig. 1a). In agreement with these published studies, GFP-Gag bearing the R50A or G110V or Δdim mutation accumulated in the nucleus (Fig. 3c, left panel). When any of these mutations was combined with the R540A substitution, the resulting GFP-Gag variants were distributed in the cytoplasm and the nucleoplasm and also accumulated in the nucleolus, to a variable extent (Fig. 3c, left panel).
Having previously shown that deletion of the CBM impairs FV Gag binding to mitotic chromosomes , we also addressed the involvement of the conserved residues within the GRII box in the interaction of FV Gag with chromatin. Gag variants where Y537 or R542 are changed to A retained the ability to interact with mitotic chromosomes (Fig. 3a, c, right panel). Notably, mutation of R540 to A was sufficient to abolish binding of either the CTD or full length Gag expressed as GFP-fusion proteins to chromatin (Fig. 3a, c, right panel). The results obtained in metaphase spreads were confirmed by the observation of fixed cells expressing GFP-GRs R540A that undergo mitosis, as judged by DNA staining (Fig. 3b). Similarly, we never observed an association between mitotic chromosomes and GFP-GRs bearing the R540F mutation, which mimics constitutive methylation (Fig. 3b). Finally, we found that GFP-GRs carrying the R540 K change painted the chromosomes of mitotic cells (Fig. 3b), indicating that a positive charge at position 540 is sufficient to ensure interaction of Gag with the H2A/H2B core histones [19, 21]. Altogether our findings indicate that the phylogenetically conserved R540 residue of PFV Gag is critical to regulate the subnuclear distribution of the protein: i.e. its nucleolar localization versus mitotic chromosomes binding.
R540 is required for both PRMT1 binding and ADMA modification of PFV Gag
PRMT1 is the primary methyltransferase that deposits the asymmetric dimethylarginine (ADMA) mark, whereas PRMT5 performs the symmetric dimethylarginine (SDMA) modification. In GFP-PRMT1-expressing cells WT Gag, but not the R540A mutant, can be precipitated with an antibody specific for the ADMA modification (Fig. 4a, IP ADMA). Similarly, WTHA-GRs, but not the R540A mutant, was enriched on beads coated with the anti-ADMA antibody (Fig. 4b). In contrast, both WT Gag and the R540A mutant co-precipitated with an anti-SDMA antibody, when expressed together with GFP-PRMT5 (Additional file 3: Fig. S3B).
Since mutation of R540 also leads to the accumulation of Gag in nucleoli, we finally asked whether PRMT1 might influence the subcellular distribution of the viral protein. To this purpose, we studied the localization of GFP-GRs in HeLa cells previously transfected with siRNA targeting PRMT1 or the appropriate scrambled control. As shown in Fig. 4c, GFP-GRs is distributed throughout the nucleoplasm of control cells, while accumulates in nucleoli upon siRNA-mediated knock-down of PRMT1. Altogether these results indicate that PRMT1-dependent methylation of PFV Gag C-terminal region requires the invariant R540 residue and is necessary to prevent Gag accumulation in nucleoli.
It is well established that incoming FV Gag tethers the PIC to host cell chromatin contributing to integration site selection [19, 20, 21]. We also showed that PFV Gag harbors a NES, which integrity is required for the completion of the late stages of viral replication . These observations suggest that Gag transits through the nucleus at a step following its translation although the mechanisms underlying its ability to cross the nuclear membrane are still unclear . To get further insights in the role of Gag nuclear trafficking for FV replication, we investigated the localization of the C-terminal GR boxes and found that GRI and GRIII act as NoLSs able to target a heterologous protein (GFP) to the nucleolus. This observation underscores once more the functional link between these two motifs . We also provide evidence that PFV Gag accumulates to nucleoli in a context of infection using two complementary approaches. First, we demonstrated that Gag binds to and, at least partially, colocalizes with a Gag-TRAP-GFP decoy constitutively localized in the nucleolus of PFV-infected cells. Second, we visualized Gag in the nucleolus of PFV-infected cells exposed to conditions that slow down protein trafficking (hypoxia and/or LMB treatment), indicating that nucleolar accumulation occurs during the viral cycle and is not a mere artifact of Gag-TRAP-GFP expression. Our findings complement previous reports that Gag and/or the isolated NC proteins from several retroviruses display nuclear/nucleolar distribution, when expressed either as individual proteins or during viral infection (reviewed in ). The importance of this nuclear/nucleolar stage for retroviral replication is still unknown. Notably, the observation that many proteins of RNA viruses involved in genome packaging and viral particle assembly localize to nucleoli [44, 45] suggests that these nuclear bodies could be sites where viral ribonucleoprotein complexes form to facilitate viral RNA export and packaging .
Another major finding of our work is that neither full-length Gag nor an N-terminal truncation mutant encompassing the three GR boxes accumulates in nucleoli unless the entire GRII box or the CBS is deleted, leading us to assume that this region might hold determinant(s) antagonizing nucleolar targeting. We mapped this determinant to the invariant R540 residue, which mutation is sufficient to induce nucleolar targeting of the isolated GRII box or Gag CTD. Of note, localization of full-length Gag harboring the R540A mutation in nucleoli required concomitant inactivation of N-terminal motifs such as the CTRS, the NES or the dimerization domain, conditions that favor nuclear accumulation of the viral protein. Consistent with this observation, Müllers et al.  reported that PFV Gag displays nucleolar staining when fused to a heterologous NLS and upon simultaneous deletion of the GRII box. Similarly, Lochmann et al.  described nucleolar localization of RSV Gag after having enhanced its concentration in the nucleus by inhibition of CRM1-dependent nuclear export or mutation of its NES.
Having previously shown that the CBM mediates tethering of PFV Gag to chromatin , we assessed the implication of R540 in this process. Our data show that substitution of R540 to A prevents Gag from binding to mitotic chromosomes and interacting with the H2A histone. This finding is in agreement with the recent results of Lesbats et al.  demonstrating that R540 acts as an anchor motif interacting with the acidic patch on the surface of the H2A/H2B heterodimer. The role of R540 in modulating chromatin binding is further supported by the observation that insertion of the WT chromatin-binding sequence (CBS, aa 534–546) of PFV Gag, but not the corresponding R540A mutant, restores the interaction between a mutant version of MLV p12 and mitotic chromosomes . Notably, we found that Gag CTD bearing the R540 K mutation associates to chromatin in cells undergoing unperturbed mitosis, indicating that a positive charge at position 540 is necessary and sufficient to ensure tethering of PFV Gag on chromatin, but not to antagonize nucleolar accumulation.
Finally we set to investigate how R540 regulates the subnuclear localization of PFV Gag. Post-translational modification of R by methylation has been shown to modulate the function and/or localization of several viral proteins. Studies on HIV NC revealed that mutation of R residues within the NoLSs, which are targeted by PRMT6-mediated methylation, impairs both nucleolar localization  and reverse transcription initiation . Methylation is also proposed to control both the nucleolar distribution and the transactivation activity of the HIV Tat protein [49, 50]. In the case of HIV Rev mutation of methylated R residues or expression of catalytically inactive PRMT6 diminishes both binding to and nuclear export of RRE-containing transcripts . Least but not last, methylation of R residues influences histone binding of KSHV (Kaposi Sarcoma-associated Herpesvirus) LANA (Latency-associated Nuclear Antigen) protein . Based on these reports, we asked whether PFV Gag is methylated and whether this post-translational modification might influence its subnuclear distribution. In our work we show for the first time that PFV Gag interacts with and is methylated by both PRMT1 and PRMT5. We also established that Gag R540A mutant retains the ability to interact with PRMT5 and, surprisingly, displays enhanced SDMA modification compared to WT Gag. Why mutation of the R540 residue would facilitate deposition of SDMA marks by PRMT5 and at which sites this modification occurs are currently unanswered questions. Importantly, substitution of R540 with A abolished both Gag association with, and modification by, PRMT1. In addition, PFV Gag C-terminus fused to GFP (GFP-GRs) is enriched in nucleoli when PRMT1 expression is reduced by RNA interference, mimicking the phenotype of the R540 mutation. These data are consistent with a model according to which PRMT1-mediated modification of PFV Gag antagonize its nucleolar accumulation. Although we do not provide direct evidence of PRMT1-mediated methylation of R540, it is tempting to speculate that reversible modification of this amino acid might contribute to finely tune the distinct functions of Gag at different stages of the replication cycle. Nevertheless, finding that Gag mutants where R540 is mutated to A or F, which mimics constitutive methylation [53, 54], have a similar phenotype argues that methylation at this site is neither required for localization in nucleoli nor for tethering to chromatin. Another possibility is that PRMT1 controls Gag subnuclear localization by mediating ADMA modification of other residues within its R-rich C-terminal region, which await identification. PFV Gag was already known to be phosphorylated on multiple sites [5, 9]. Recent studies established that phosphorylation of T225 occurs exclusively in virions and propose that this modification is required for the interaction between PFV Gag and Polo-like kinases, ultimately leading to efficient integration .
When we assessed the impact of nucleolar retention of Gag on PFV replication we found that virions produced from Gag-TRAP-GFP-expressing cells display only a moderate decrease of infectivity, which is not statistically significant as compared to the infectivity of virions released from cells expressing Gag1-200-GFP, leaving the question of the role of PRMT1-dependent methylation and/or nucleolar accumulation of Gag during PFV replication open for further investigations. Given that a virus harboring the R540Q mutation within the CBM has an altered integration profile , it would also be interesting to address the role of Gag methylation for integration site selection.
In closing, our work underscores that Gag localizes in the nucleolus during PFV replication. This step is likely regulated by PRMT1-mediated methylation of Gag that depends on the invariant R540 residue. Further studies will be required to define the functional significance of the nucleolar step for FV replication as well as the consequences of PFV Gag methylation in regard to the regulation of its complex nuclear transport and integration site selection.
Cells and culture conditions
HeLa, 293T and U373MG cells were cultured in DMEM supplemented with 10% Fetal Calf serum (FBS). BHK-U3GFP indicator FAG (Fluorescence Activated GFP) cells were cultured in DMEM supplemented with 5% FBS and 500 µg/mL G418 (Gibco). Leptomycin B (LMB) (Sigma) was added to culture medium to a final concentration of 10 nM for 4 h. Hypoxic conditions (2% O2, 5%CO2 and 93% N2) were induced by a continuous flow of nitrogen using a Forma Series II Water Jacket CO2 incubator (model: 3131; Thermo Scientific).
Fusion of individual GR boxes (GRI: aa 485–511, GRII: aa 534-557, GRIII: aa 586–618) or RevNoLS (aa 35–51) to GFP or RFP was obtained by inserting annealed complementary oligonucleotides of appropriate sequence into pEGFP-C1 or pDsRed-C1. GFP-GRs (GRs: aa 477–625) and GFP-Gag were constructed by insertion of PCR products obtained using the pcziGag as template, into pEGFP-C1 (Clontech) between HindIII and BamHI sites. HA-GRs expression plasmids were generated by replacing GFP by the HA sequence. pHFVGag∆131-162  served as template to generate GFP-Gag∆dim (deletion of PFV Gag aa 130–160). Mutants were generated using QuickChange site-directed mutagenesis Kit according to the manufacturer’s specifications (Stratagene). Fragments spanning Gag1-200, RevNoLS or Gag1-200 fused to RevNoLS, were inserted into pEGFP-N1, and the resulting plasmids were used as template to amplify the coding sequences to be inserted in pMSCVneo at the EcoRI and HpaI sites. Plasmids encoding GFP-tagged human PRMTs proteins were kindly provided by Mark Bedford .
Establishment of cell lines stably expressing Gag-TRAP-GFP
U373MG stable cell lines were established using the Murine Stem Cell Virus (MSCV)-based retroviral vector system. Recombinant retroviral vectors were generated by transfection of 293T cells with the pMSCV-neo vector encoding Gag1-200-RevNoLS-GFP (Gag-TRAP-GFP), Gag1-200-GFP, RevNoLS-GFP or GFP, and the packaging plasmids expressing MLV Gag-Pol and the Vesicular Stomatitis Virus envelope G glycoprotein (VSV-G) using the calcium phosphate precipitation method. Cell-free supernatants were collected 48 h post-transfection and used to transduce U373MG cells. GFP expression was analyzed 48 h post-transduction by flow cytometry. After cell sorting, GFP-positive cells were propagated in culture medium supplemented with G418 (500 µg/mL).
Indirect immunofluorescence imaging on fixed-cells or mitotic chromosome spreads was described elsewhere . In brief, samples were incubated with the appropriate primary antibodies (4 °C, overnight) and fluorescent-labeled secondary antibody (30 min, room temperature). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired with a laser-scanning confocal microscope (LSM510 Meta; Carl Zeiss) equipped with an Axiovert 200 M inverted microscope, using a Plan Apo 63_/1.4-N oil immersion objective.
Co-immunoprecipitation assay and Western blotting
Cell pellets were lysed in 0.4 M NaCl, 1 mM MgCl2, 10% sucrose, 0.5 mM DTT, 10 mM PIPES pH 6.8, 0.5% NP-40 supplemented with Protease Inhibitor Cocktail (Roche) (30 min on ice), and subsequently centrifuged (12,000g, 5 min at 4 °C). Immunoprecipitation and Western-blot were performed as previously described .
Transfected 293T cells were prepared as described . After extensive washing, the grids were incubated with an anti-GFP monoclonal antibody (90 min, RT), followed by an anti-rabbit antibody conjugated to 15 nm-gold particles (British Biocell International, Cardiff, UK) (90 min, RT). Ultrathin sections were stained with 5% uranyl acetate 5% lead citrate, placed on EM grids coated with collodion membrane and observed with a Jeol 1010 transmission electron microscope (Tokyo, Japan).
HeLa Cells were transiently transfected with ON-TARGETplus Human SMARTpool siRNA targeting PRMT-1 (Dharmacon #3276) or the scrambled control (10 nM) using the Lipofectamine RNAiMax reagent according to the manufacturer’s instructions (Life Technologies). After 48 h, cells were transfected with the GFP-GRs expressor and, following further 24 h incubation, were fixed and analyzed by confocal microscopy.
Graphical representation and statistical analyses were performed using the GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Differences were tested for statistical significance using ne-way ANOVA statistical test with a Bonferroni Multiple comparison post-test.
AS, JP, JTT conceived and designed the experiments; JP, JG, JTT, MLG, and PR performed the experimental work; AS, AZ, FB, JP, JTT, MLG, PL analyzed the data; JTT, AS and AZ wrote the manuscript. All authors read and approved the final manuscript.
We thank Axel Rethwilm and Dirk Lindemann for FV reagents; Mark Bedford for GFP-PRMTs plasmids; Christelle Doliger, Sophie Duchez and Niclas Setterblad at the Imaging, Cell selection and Genomics Department of the Institut Universitaire d’Hématologie for confocal microscopy; Claudine Pique for critical reading of the manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The dataset used and/or analyzed during the current study are available from the corresponding author on a reasonable request.
This study was supported by CNRS, INSERM, Université Paris Diderot Sorbonne Paris Cité, CNAM and ANR (ANR-12-BSV3-0016 to AS, ANR-15-CE15-0008 to AS and FB).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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