Docking simulation predicts that mCRP binds to integrin αvβ3 but pCRP does not.

<p>a) The headpiece of integrin αvβ3 (PDB code 1LG5) was used as a target. The docking model predicts that mCRP (red) binds to the RGD-binding site of the integrin αvβ3 headpiece (green and blue). Amino acid residues involved in αvβ3-mCRP interaction are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093738#pone-0093738-t001" target="_blank">Table 1</a>. Cations (Mn) and cyclic RGD peptide and specificity loop of β3 are close to the predicted mCRP-binding site in integrin αvβ3. Cations and cyclic RGD peptide in 1LG5 were removed during docking simulation. The predicted integrin-binding site in mCRP is also close to the phosphocholine-binding site and the RQD motif in mCRP. b) To check if pCRP binds to the integrin, we superposed the pentameric CRP (pCRP, orange and red) to the bound mCRP (red). Interestingly, there are steric clashes between pentameric CRP and αvβ3. This predicts that pentameric CRP can not fully access to mCRP-binding site in integrins due to steric hindrance.</p

mCRP binds to U937 monocytic cells, and induces robust chemotaxis in an integrin-dependent manner.

<p>a).<u> Adhesion of U937 cells to CRP isoforms</u>. Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. Wells were incubated with U937 cells (10⁵ cells per well) for 1 h in RPMI1640 and bound cells were quantified. b) and c). <u>Effect of antagonists to αvβ3 and α4β1 on adhesion of U937 cells to mCRP</u>. In b), 2.5 μM coating concentration of mCRP was used. Antibodies used were mAb 7E3 (to human β3, 25 μg/ml), mAb SG73 (to human α4, 25 μg/ml), and AIIB2 (to human β1, 25 μg/ml). “mIgG” represents purified mouse IgG used as a control. Antagonists used were cyclic RGDfV (to αvβ3, 10 μM) and BIO1211 (to α4β1, 1 μM). DMSO was used as a control. Adhesion assay was performed in RPMI. d) mCRP induces AKT activation in U937 cells, but not ERK1/2 activation. U937 cells were serum-starved and stimulated with pCRP and mCRP (100 μg/ml) and cell lysates were analyzed by western blotting. d) <u>mCRP, and less effectively pCRP, induce chemotaxis of U937 cells in an integrin-dependent manner.</u> Chemotaxis was measured in modified Boyden chambers (Transwells). 50 μg/ml mCRP or pCRP in 600 μl RPMI 1640 medium was placed in the lower chamber, and U937 cells (5×10⁵ cells per well) were placed in the upper chamber. U937 cells were preincubated with antibodies (25 μg/ml) for 30 min at 37°C. After 4 h incubation, migrated cells were counted. e) A PI3K inhibitor, not MEK inhibitor, suppresses mCRP-induced chemotaxis of U937 cells. LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) were added at 50 μM in the chemotaxis medium.</p

Amino acid residues involved in mCRP-αvβ3 interaction.

<p>Amino acid residues in integrin αvβ3 and mCRP within 6 Å to each other in the docking model were identified using Swiss-pdb viewer v. 4.1.</p

mCRP specifically binds to αvβ3, but pCRP less efficiently binds to the integrin.

<p>Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. a<u>) ELISA-type integrin binding assay.</u> In a) wells were incubated with recombinant soluble αvβ3 for 2 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl₂. b) <u>Effect of heat treatment on mCRP binding to soluble αvβ3.</u> We heated (90°C for 20 min) mCRP before coating wells and used for binding assays. Assays was performed as in a). c) <u>β3- and β1-3-1-CHO cells adhere to mCRP, but β1-CHO cells did not adhere well to mCRP.</u> mCRP was incubated with β1-CHO, β3-CHO, or β1-3-1-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl₂. Bound cells were quantified. d) <u>Specificity of αvβ3 binding to mCRP.</u> We tested if inhibitors of αvβ3 block adhesion of β3-CHO cells to mCRP. mCRP was incubated with cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl₂. mAb 7E3 (to human β3, 10 μg/ml) and cyclic RGDfV (specific antagonist to αvβ3, 10 μM) blocked the adhesion of β3-CHO cells to mCRP, but control purified mouse IgG (mIgG) or vehicle DMSO did not. e) <u>Cation dependency of mCRP binding to αvβ3</u>. Adhesion assays were performed as described in c). mCRP was incubated with β3-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 2 mM cations or EDTA. The coating concentration of mCRP is 50 μg/ml. The levels of adhesion in different cation conditions are statistically different. f) Alignment of β1, β3, and β1-3-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093738#pone.0093738-Takagi1" target="_blank">[17]</a>. g) <u>Specificity of β1-3-1 integrin binding to mCRP.</u> We tested if anti-human β1 mAb AIIB2 blocks β1-3-1-CHO cells adhesion to mCRP. (Note: 99% of β1-3-1 is β1 and mAb AIIB2 binds to β1-3-1 and blocks its function). h) <u>Cell adhesion to pCRP. </u>pCRP was incubated with β1-CHO, β3-CHO, or β1-3-1-CHO cells for 1 h at 37°C in Tyrode-HEPES buffer with 1 mM MgCl₂. Bound cells were quantified.</p

mCRP, and less efficiently pCRP, specifically bind to α4β1.

<p>a). Adhesion of α4-CHO cells to CRP isoforms. Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. Wells were incubated with CHO cells that express recombinant α4β1 (α4-CHO, 10⁵ cells per well) for 1 h in Tyrode-HEPES buffer with 1 mM MgCl₂ and bound cells were quantified. b). Effect of antagonists to α4β1 on adhesion of α4-CHO cells to mCRP. Experiments were performed as in a). Fifty μg/ml coating concentration of mCRP was used. Antagonists were mAb SG73 (anti-human α4, 10 μg/ml) and BIO1211 (specific antagonist to α4β1, 1 μM). “mIgG” represents purified mouse IgG used as a control.</p

Biphasic euchromatin-to-heterochromatin transition on the KSHV genome following <i>de novo</i> infection.

<p>As soon as the viral genome enters the nucleus, histones are recruited on the viral DNA, resulting in the chromatinization of the KSHV genome. The viral genome initially adopts a transcriptionally permissive chromatin, characterized by high levels of the H3K27ac and H3K4me3 and this is accompanied by the transient expression of lytic genes. Subsequently, the PRC2 and PRC1 are recruited to viral genome, where they are responsible for the enrichment of H3K27me3 and H2Ak119ub on the viral chromatin as well as the repression of lytic genes. During latency, both PRC2 and PRC1 remain on the KSHV genome for the maintenance of the repression of lytic genes.</p

<i>De novo</i> chromatinization of the KSHV genome following infection.

<p>(<b>A</b>) FAIRE assay showing the degree of chromatinization of the indicated viral and cellular promoters in SLK cells infected for 1, 8 or 24 hours or in latently infected SLK cells. (<b>B</b>) and (<b>C</b>) The enrichment of histones H3 and H2A was calculated as ChIP/input (% Input or occupancy) at the indicated viral and cellular promoters following <i>de novo</i> infection at 1, 8 and 24 hpi and in latently infected SLK cells. (<b>D</b>) Real time RT-PCR analysis was performed to determine the expression of the indicated viral genes in SLK cells infected by KSHV for 8 and 24 hours and in latently infected SLK cells. Expression levels of viral genes are shown relative to those of 18S. A 2-tailed student's t-test was performed between 8 hpi and latency for each tested lytic gene and all p values were less than 0.05.</p

Analysis of the deposition of histone modifications on KSHV promoters during <i>de novo</i> infection using time-course ChIP assays.

<p>ChIPs were performed using latently infected SLK cells or SLK cells following <i>de novo</i> infection for the indicated hours post infection (hpi) and the enrichment of histone modifications was measured by qPCR using primers specific for the indicated viral and cellular promoters. ChIPs for each histone modification were normalized for the amount of the relevant histones at each promoter. T-test was applied to compare the values between the indicated time points (*). (<b>A</b>) H3K4me3 ChIP (for LANA, RTA and K2 p<0.05, for ORF25 p = 0.1 between 1 and 24 hpi). (<b>B</b>) H3K27ac ChIP (for LANA p = 0.6, RTA and K2 p<0.05, for ORF25 p = 0.18 between 1 and 72 hpi). (<b>C</b>) H3K27me1 ChIP (for LANA p = 0.063, for RTA, K2 and ORF25 p<0.05 between 1 and 8 hpi). (<b>D</b>) H3K27me3 ChIP (for LANA p = 0.33, for RTA, K2 and ORF25 p<0.005 between 1 and 72 hpi). (<b>E</b>) H2AK119ub ChIP (for LANA p = 0.3, for RTA, K2 and ORF25 p<0.02 between 1 and 72 hpi). (<b>F</b>) Sequential deposition of H3K27ac and H3K27me1 on viral promoters during infection was confirmed by sequential ChIP assays. The first ChIP was performed with H3K27ac-specific antibody at 1 hpi and 8 hpi, followed by elution of the immunoprecipitated DNA, The eluted DNA was used as the input for a second ChIP performed with H3K27me1 antibody. (<b>G</b>) Sequential deposition and colocalization of H3K4me3 and H3K27me3 on the RTA promoter following <i>de novo</i> infection were confirmed by sequential ChIP assays. The first ChIP was performed with H3K4me3-specific antibody at 24 hpi and 72 hpi followed by the second ChIP for H3K27me3 using the eluted first ChIPs as the input.</p

Euchromatinization of the KSHV genome in gingival oral epithelial cells following <i>de novo</i> infection.

<p>(<b>A</b>) Measurement of viral DNA replication in SLK, OEPI, SCC15 and NOK cells infected by KSHV for 4, 24, 48 and 72 hours. The viral DNA polymerase inhibitor, PAA, was applied to block the replication of KSHV. (<b>B</b>) FAIRE assay showing the degree of chromatinization of the indicated viral and cellular promoters in SLK and OEPI cells infected for 8, 24 or 72 hours. (<b>C</b>) SLK and OEPI cells were infected with KSHV for 1, 2 and 3 days and immunoblots were performed to test the expression of RTA and K3 viral proteins. Actin served as a loading control. The “C” indicates immunoblot analysis of uninfected cells. (<b>D</b>) Quantitative RT-PCR analysis of viral gene expression in KSHV infected SLK and OPEI cells. (<b>E</b>) and (<b>F</b>) ChIP analysis of the indicated histone modifications on a selection of KSHV promoters in OEPI cells at 8, 24 and 72 hpi. The cellular promoters (ACT and MYT1) were used as controls. (<b>G</b>) Comparative immunoblot analysis of the indicated cellular proteins between SLK and oral epithelial cells.</p

RTA is involved in the deposition of activating histone marks on the KSHV genome following <i>de novo</i> infection.

<p>(<b>A</b>) Comparative ChIP analysis of H3K4me3, H3K27ac and H3K27me3 at the indicated viral promoters of wild type (wt) or RTA knockout (RTAstop) KSHV-infected SLK cells at 8, 24 and 72 hpi. The promoters of the cellular actin (ACT) and MYT1 genes were used as controls. ChIPs were normalized for the amount of histone H3 at each promoter. The asterisk denotes p<0.05 between wt and RTAstop at 8 hpi. (<b>B</b>) RT-qPCR analysis of viral gene expression in RTAstop KSHV-infected SLK cells at 24 hpi. The expression of the indicated viral genes was calculated relative to wt KSHV-infected cells. (<b>C</b>) ChIP experiments showing the binding of RTA and CBP on the RTA-responsive RTA and K2 promoters as well as the LANA promoter in latently-infected cells and naïve cells infected for 8 or 24 hours by wild type KSHV. ORF25 promoter was used as a negative control. (<b>D</b>) RTA and CBP binding on viral promoters in RTAstop KSHV-infected SLK cells.</p