Gammaretroviruses tether to mitotic chromatin by directly binding nucleosomal histone proteins

The gammaretroviral gag cleavage product, p12, is essential for replication at both early and late stages of the virus life cycle. During the early stage of infection, the viral core is released into the cytoplasm, the viral RNA genome is reversed transcribed to cDNA and this viral DNA is then integrated into the host cell chromatin to form a provirus. The p12 protein has N- and C-terminal domains (NTD and CTD) that are required for steps leading up to integration, but the molecular details of their functions remain poorly characterised. Using the prototypic gammaretrovirus, murine leukemia virus (MLV) as a model, we recently showed that the NTD of p12 directly binds to and stabilises the capsid (CA) lattice of the viral core. Alterations to the CTD of MLV p12 prevented the viral pre-integration complex (PIC) tethering to host chromatin in mitosis, and this could be partially rescued by addition of a heterologous chromatin binding motif. In this study we demonstrated that the CTD of p12 directly binds to nucleosomal histone proteins, targeting not only p12 but also CA to mitotic chromatin. Additionally, cell-cycle-dependent phosphorylation of p12 appeared to increase the affinity of p12 for chromatin in mitosis relative to interphase. Thus, we have revealed how p12 can link the CA-containing PIC to mitotic chromatin, ready for integration. Importantly, we observed that direct binding to nucleosomes is a conserved feature of p12 orthologs across the gammaretrovirus genus and that the nucleosomal docking site is potentially shared with that of spumaretroviral Gag proteins

New antigens for multi-component blood-stage vaccines against Plasmodium falciparum malaria

This dataset is from a prospective longitudinal cohort study of children from Chonyi village in Kilifi County, Kenya. The study was conducted for the purpose of evaluating the potential protective efficacy of antibodies to Plasmodium falciparum antigens against clinical episodes of malaria. Venous blood was collected from 286 children (age 1-10 years old) during a cross-sectional survey at the start of a malaria transmission season. Thick and thin blood smears were prepared for parasite detection by microscopy. Plasma was used to measure antibody levels to 39 Plasmodium falciparum merozoite surface and secreted proteins by enzyme linked immunosorbent assay. The children were then followed up for a period of 6 months with both active (Weekly home visits) and passive surveillance for malaria case detection.</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-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

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

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