Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

<div><p>The murine leukaemia virus (MLV) Gag cleavage product, p12, is essential for both early and late steps in viral replication. The N-terminal domain of p12 binds directly to capsid (CA) and stabilises the mature viral core, whereas defects in the C-terminal domain (CTD) of p12 can be rescued by addition of heterologous chromatin binding sequences (CBSs). We and others hypothesised that p12 tethers the pre-integration complex (PIC) to host chromatin ready for integration. Using confocal microscopy, we have observed for the first time that CA localises to mitotic chromatin in infected cells in a p12-dependent manner. GST-tagged p12 alone, however, did not localise to chromatin and mass-spectrometry analysis of its interactions identified only proteins known to bind the p12 region of Gag. Surprisingly, the ability to interact with chromatin was conferred by a single amino acid change, M63I, in the p12 CTD. Interestingly, GST-p12_M63I showed increased phosphorylation in mitosis relative to interphase, which correlated with an increased interaction with mitotic chromatin. Mass-spectrometry analysis of GST-p12_M63I revealed nucleosomal histones as primary interactants. Direct binding of MLV p12_M63I peptides to histones was confirmed by biolayer-interferometry (BLI) assays using highly-avid recombinant poly-nucleosomal arrays. Excitingly, using this method, we also observed binding between MLV p12_WT and nucleosomes. Nucleosome binding was additionally detected with p12 orthologs from feline and gibbon ape leukemia viruses using both pull-down and BLI assays, indicating that this a common feature of gammaretroviral p12 proteins. Importantly, p12 peptides were able to block the binding of the prototypic foamy virus CBS to nucleosomes and <i>vice versa</i>, implying that their docking sites overlap and suggesting a conserved mode of chromatin tethering for different retroviral genera. We propose that p12 is acting in a similar capacity to CPSF6 in HIV-1 infection by facilitating initial chromatin targeting of CA-containing PICs prior to integration.</p></div

GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated.

<p>(A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of <i>LacZ</i>-encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of >3 biological replicates).</p

GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS.

<p>Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log₂(L/M) silac ratios of the set of MS hits (FDR <5%) from each replicate were plotted as a frequency distribution. Mean and standard deviation (SD) of each distribution was estimated by fitting to a normal distribution curve (R²≥0.91). MS hits with log₂(L/M) ratios greater than 2.58 SDs from the mean were selected as significantly enriched in the GST-p12_M63I (L) sample. Of the 314 such proteins identified in R1, 68 were also found in R2 (Venn diagram). (B) Functional classification of the 68 GST-p12_M63I interacting proteins in (A) was performed based on InterPro domain annotation. (C) The abundance factor for each protein was calculated by dividing the peptide spectral count by its length. These values were then normalised across the group and the scores for R1 plotted against the scores for R2 (Pearson correlation <i>r</i> = 0.99). MS hits with normalised abundance factors ≥1 in both replicates are boxed in red (top panel) and expanded in the bottom panel. The points are coloured according to the protein function shown in the key: Blue, nucleosomal histone proteins; Red, chromobox homolog proteins; Green, chromosomal passenger complex proteins; Black, others. (D) Validation of SILAC results. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1), p12_mut14 (lane 2), p12_M63I (lane 3), p12_M63I/mut14 (lane 4), p12+<i>h</i>CBS (lane 5) and p12+<i>h</i>CBS/mut14 (lane 6) and GST pull-down assays were performed and analysed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007117#ppat.1007117.g006" target="_blank">Fig 6</a>. Immunoblots (right hand panels) were probed with a selection of antibodies to host proteins of interest (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007117#ppat.1007117.s008" target="_blank">S4 Table</a>). Bands corresponding to core histones in the silver-stained gel are starred.</p

Identifying phosphorylation sites of GST-p12 precipitated from 293T cells using nano LC–MS/MS.

<p>Identifying phosphorylation sites of GST-p12 precipitated from 293T cells using nano LC–MS/MS.</p

GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis.

<p>(A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+<i>h</i>CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+<i>h</i>CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+<i>h</i>CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+<i>h</i>CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic <i>versus</i> interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.</p

GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin.

<p>(A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+<i>h</i>CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of <i>LacZ</i>-encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+<i>h</i>CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of >3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007117#ppat.1007117.g009" target="_blank">Fig 9</a>) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.</p

The p12 CTD directly binds recombinant nucleosomal arrays.

<p>Direct interactions between p12 CTD peptides and nucleosomal histone proteins was tested using biolayer interferometry (BIL). Streptavidin sensor probes were coated with biotinylated p12 CTD or <i>h</i>CBS (positive control) peptides to equivalent levels and then immersed in analyte solutions containing recombinant poly-nucleosomes to measure binding. (A) Representative sensorgrams showing binding of peptides corresponding to the CTD of Mo-MLV p12_M63I, <i>phos</i> p12-M63I (S61-phosphorylated), p12_M63I/R66A or <i>h</i>CBS to recombinant poly-nucleosomes (~250 nM). Black arrows indicate the beginning and end of the association phase. (B-D) Affinity measurements were derived by probing peptides corresponding to <i>h</i>CBS (B-D) or the CTD of Mo-MLV p12_M63I, <i>phos</i> p12-M63I (B), GaLV p12, FeLV p12 (C) or Mo-MLV p12_WT (D) against a range of serially-diluted nucleosomes. Equilibrium binding data from two technical replicates were pooled for the analysis. The plotted data are fractional saturation of binding as a function of nucleosome concentration. (E) Competition assay inhibiting nucleosome binding to immobilised biotin-tagged <i>h</i>CBS with soluble non-biotinylated <i>h</i>CBS or Mo-MLV p12 CTD peptides as indicated in key (100 μM). Left panel shows representative sensorgrams with black arrows indicating the beginning and end of the association phase. The bar chart shows equilibrium binding of <i>h</i>CBS in the presence and absence of competing peptides as mean ± SD from three technical replicates. (F) Representative sensorgrams showing competition assay inhibiting nucleosome binding to immobilised biotin-tagged p12 CTD peptides or <i>h</i>CBS, as indicated in graph title, with soluble non-biotinylated <i>h</i>CBS (100 μM, red lines). Black arrows indicate the beginning and end of the association phase. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007117#sec015" target="_blank">methods</a> for peptide sequences.</p

GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag.

<p>Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log₂(H/L) silac ratios of the set of MS hits (FDR <5%) from each replicate (R1 and R2) were plotted as a frequency distribution. Mean and standard deviation (SD) of each distribution was estimated by fitting to a normal distribution curve (R² ≥ 0.98). MS hits with log₂(H/L) ratios greater than 2.58 SDs from the mean were selected as significantly enriched in the GST-p12_WT (H) sample. Of the 21 such proteins identified in R1, 7 were also found in R2 (Venn diagram), and were investigated further. (C) Ranking of the 7 potential GST-p12_WT binding proteins. The abundance factor for each protein was calculated by dividing the peptide spectral count by its length. These values were then normalised across the group and the scores for R1 plotted against the scores for R2. The points are coloured according to the protein function shown in the pie chart. CLTC = clathrin heavy chain, CLINT1 = clathrin interactor 1, WWP2 = NEDD4-like E3 ubiquitin ligase, CLTA = clathrin light chain, PI3KC2A = phosphatidylinositol-4-phosphate-3-kinase C2, SEC16A = protein transport protein Sec16A, INCENP = inner centromere protein. (D) The relative enrichment of the 7 proteins (putative GST-p12_WT interactors) in the GST-p12_WT (H) samples compared to GST-p12_mut14 (M) samples was estimated from the log₂(H/M) ratios. (E) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to selected host proteins. Mutant nomenclature is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007117#ppat.1007117.g001" target="_blank">Fig 1</a>. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12 mutants (lanes 3–10) or GST (lane 1) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-CLINT1, anti-WWP2 and anti-H2B antibodies.</p

Models for p12-chromatin binding.

<p>(A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.</p

The N-terminal domain of MLV p12 directly binds hexameric CA arrays.

<p>(A) Schematic representation of the MLV Gag-Pol expression plasmid used in this study, showing the Mo-MLV p12 sequence with alanine substitution mutants below. (B) Immunoblot showing binding of p12 to N-MLV CA. His-tagged WT (lane 2) or P1G mutant (lane 3) CA proteins were immobilised on lipid nanotubes comprising the Ni^2+-chelating lipid, DGS-NTA, prior to incubation with purified p12_WT, p12_mut6 (NTD mutant) or p12_mut14 (CTD mutant). No lipid tubes or CA were included in the negative control reactions (lane 4). CA nanotubes were pelleted, and any bound p12 was revealed by SDS-PAGE and immunoblotting with a rabbit anti-p12 antibody. 1/40^th of the purified p12 protein input was loaded in lane 1. (C) Co-immunoprecipitation (Co-IP) of CA with p12 from viral lysates. Mo-MLV virus-like particles (VLPs) carrying myc-tagged p12_WT (lanes 1 and 2), p12_mut6 (lanes 3 and 4) or p12_mut14 (lanes 5 and 6) were either untreated (lanes 1, 3 and 5) or fixed in 1% formaldehyde (lanes 2, 4 and 6) prior to lysis. Myc-p12 was immunoprecipitated from normalised viral lysates using an anti-myc antibody. 1/50^th of input lysates and 1/5^th of Co-IP elutions were analysed by immunoblotting with anti-CA and rabbit anti-p12 antibodies. (D) Immunoblot showing binding of p12 specifically to CA hexamers. His-tagged WT (lanes 2 and 4) or P1G mutant (lanes 3 and 5) CA proteins were immobilised as hexamers on lipid nanotubes (lanes 2 and 3), or randomly on Ni-NTA beads (lanes 4 and 5). No CA, beads or tubes were included in the negative control (lane 6). After incubation with p12, the CA assemblies were pelleted and analysed by SDS-PAGE and immunoblotting for CA (anti-His) and p12 (mouse anti-p12). 1/40^th of the purified p12 protein input was loaded in lane 1.</p