The invariant arginine within the chromatin-binding motif regulates both nucleolar localization and chromatin binding of Foamy virus Gag

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Keywords: Chromatin-binding; Foamy virus; Gag; Methylation; Nuclear trafficking; Nucleolus; PRMT; Post-translational modification.

Fig. 1

GRI and GRIII boxes of PFV Gag are Nucleolar Localization Signals. a Scheme of PFV Gag protein where the primary protease-cleavage site at residue 621 is indicated by a dotted line. Some characterized motifs are shown. CTRS: cytoplasmic targeting and retention signal (aa 43–60); NES: nuclear export signal (aa 95–112); dim: dimerization domain (aa 130–160); GRI, GRII and GRIII: glycine-arginine rich box I (aa 485–511), II (aa 534–557) and III (aa 586–618); CBM, chromatin-binding motif (aa 536–544). b The subcellular localization of GRI, GRII, GRII_R540A or GRIII expressed as GFP-fusion proteins in fixed HeLa cells was analyzed 24 h post-transfection by immunofluorescence and confocal microscopy. Nucleoli were immune-stained with an anti-nucleolin antibody (ab 22758, Abcam, 1:800) and nuclei were stained with DAPI (blue). c The localization of GFP-GRI expressed in HeLa cells relative to the NoLS of HIV-1 Rev protein (aa 35–51) in fusion with DsRed or specific markers of the nucleolar subcompartments was studied as in B. Cells were stained with antibodies against fibrillarin (c13c3, Cell signaling, 1:200), B23 (sc6013_R, Santa Cruz, 1:200) or UBF (H300, Santa Cruz, 1:200) to visualize the dense fibrillar component (DFC), the granular component (GC) and the fibrillar center (FC), respectively. The right column (zoom × 16) corresponds to the enlarged images from the boxed areas. Scale bar represents 10 µm

Fig. 2

Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag_1-200-RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag_1-200-GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit polyclonal antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. 1b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (*p < 0.05; **p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O₂, 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. 1b. Scale bar represents 10 µm

Fig. 3

The invariant R540 residue in PFV Gag regulates nucleolar localization and binding to mitotic chromosomes. Living HeLa cells expressing the indicated GFP-GRs (a) or GFP-Gag (c) constructs and stained with Hoechst 33342 were observed on a confocal microscope 24 h after transfection (left panels). Merged images correspond to GFP, nucleic acid staining and differential interference contrast to visualize the cell shape. The “% nucleolar” column indicates the percentage of transfected cells displaying GFP staining in the nucleolus (−, < 1%; +, 1–25%; ++, 26–50%; +++, 51–75%; ++++ , 76–100%). To study the interaction of GFP-fusion proteins with chromatin (right panels), cells ectopically expressing indicated proteins were arrested in metaphase by treatment with colcemid (0.1 µg/mL, 2 h) and chromosome spreads counterstained with DAPI. Images were acquired as described in Fig. 1b. The chromatin binding column indicates whether the GFP-fusion protein was exclusively localized onto chromosomes (++), both on chromosomes and throughout the cell (+), or was distributed throughout the cell and did not associate with chromosomes (−). Representative images from two independent experiments are shown. Between 100 and 120 cells were analyzed for each condition. Scale bars represent 10 µm. b HeLa cells expressing the indicated GFP-GRs were stained with DAPI. Images were acquired as described in Fig. 1b. Scale bars represent 10 µm

Fig. 4

PRMT-1 binds to and methylates PFV Gag in a manner that depends on R540. a Following lysis, cells expressing PFV Gag WT or R450A mutant and GFP-PRMT1 were incubated with protein A beads coated with either an anti-GFP (cat.11 814 460 001, Roche, 1:100) or an anti-ADMA (ab5394 (7E6), Abcam,1:100) antibody. Input and immunoprecipitated proteins were separated by SDS-PAGE and visualized by Western blotting with anti-GFP (cat.11 814 460 001, Roche, 1:1000) or rabbit polyclonal anti-PFV antibodies. b Lysates from cells expressing HA-tagged GRs or GRs-R540A were immunoprecipitated with an antibody directed against PRMT1 (Cat A300-722A, Bethyl Laboratories (Euromedex), 1:100), the ADMA modification (ab5394 (7E6), Abcam, 1:100), or the HA epitope (H11, Covance, 1:100). Input and bound proteins were analyzed as in A. c HeLa cells were transfected with siRNA targeting PRMT1 or scrambled control (scr) and, two days later, with GFP-GRs expression plasmid. After 24 h, cells were processed as indicated in Fig. 1b. Images are representative of two independent experiments. The numbers indicate the percentage of GFP-positive cells with significant nucleolar accumulation of 100 counted cells. Scale bar represents 10 µm