Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome

Abstract

Human cytomegalovirus (hCMV) immediate early 1 (IE1) protein associates with condensed chromatin of the host cell during mitosis. We have determined the structure of the chromatin-tethering domain (CTD) of IE1 bound to the nucleosome core particle, and discovered that IE1-CTD specifically interacts with the H2A-H2B acidic patch and impairs the compaction of higher-order chromatin structure. Our results suggest that IE1 loosens up the folding of host chromatin during hCMV infections.

Keywords: biochemistry; biophysics; chromatin; crystal structure; cytomegalovirus; human; nucleosome; structural biology; virus.

Figure 1.. Structure of the IE1-CTD–NCP complex.

(A) Location of the IE1-CTD binding site on the acidic patch of the nucleosomal surface. The histone octamer is shown as a surface representation colored according to electrostatic potential distribution (positive, blue; neutral, white; negative, red). DNA is shown as a cartoon colored white, and IE1-CTD is shown as a stick model. (B) A detailed view of the interaction between IE1-CTD and NCP. Histones H2A and H2B are shown in a ribbon representation superimposed with selected residues (in sticks) involved in interaction with IE1-CTD. Dashed lines indicate hydrogen bonds. An enlarged view of the region surrounding His481 of IE1-CTD is shown in an inset at the bottom of the figure. (C) Superposition of IE1-CTD and LANA. Both peptides are shown as a ribbon representation superimposed with sidechains (IE1-CTD, green; LANA, blue). (D) Structure-based alignment of IE1-CTD and LANA sequences. Residues colored in red are involved in similar interactions with the histones, and the two residues colored in magenta are engaged in IE1-CTD-specific interactions with histone H2A. DOI: http://dx.doi.org/10.7554/eLife.11911.003

Figure 1—figure supplement 1.. An omit electron density map of the bound IE1-CTD.

A stereo view of the simulated annealing (Fo-Fc) omit map showing the presence of IE1-CTD. The map is contoured at 2.5 σ level. A stick model of IE1-CTD is superimposed. DOI: http://dx.doi.org/10.7554/eLife.11911.004

Figure 2.. Comparison of protein binding modes to the acidic patch of NCP.

All NCP-binding peptides or protein segments, shown in a stick model superimposed onto a cartoon representation of the backbone, were superimposed onto the structure of NCP, shown in a surface representation colored according to electrostatic potential, of the IE1-CTD complex based on alignment of NCP structures. (A) The binding of IE1-CTD to NCP. (B) LANA (PDB id: 1ZLA). (C) RCC1 segment (PDB id: 3MVD). The green ovals indicate distinct binding zones of the acidic patch. The protruding ridge at the junction between zone I and zone II is also labeled. (D) Sir3 (PDB1d: 4KUD). (E) CENP-C (PDB id: 4X23). (F) PRC1-RING1B (PDB id: 4R8P). DOI: http://dx.doi.org/10.7554/eLife.11911.005

Figure 3.. ITC measurements of peptide-NCP binding affinities.

(A–G) Raw data and fitting curves of the integrated data for the indicated peptides and NCPs are shown together with the derived K_D values and fitting errors. DOI: http://dx.doi.org/10.7554/eLife.11911.006

Figure 3—figure supplement 1.. Binding of full-length IE1 to NCP.

Bindings of full-length IE1 (A), IE1-CTD (B), and IE1 lacking CTD (C) are measured by ITC at 150 mM NaCl concentration. DOI: http://dx.doi.org/10.7554/eLife.11911.007

Figure 4.. Influence of IE1-CTD on higher-order chromatin structure.

(A) AUC analyses showing that IE1-CTD has little effect on the folding of the 10-nm nucleosomal array. Green and black data points represent that of 10-nm nucleosomal arrays in the absence and presence of IE1-CTD, respectively. In contrast, sedimentation profile of the 30-nm chromatin fiber reconstituted in the presence of linker histone H1 (blue squares) was shifted with the addition of IE1-CTD (red dots). (B) Full-length IE1 shares the property of IE1-CTD in selectively altering the folding of the 30-nm chromatin fiber. (C) A truncation variant of IE1 lacking CTD (IE1ΔC) does not alter chromatin structure. (D–F) Indicated IE1-CTD mutants retain the ability to impact the folding of the 30-nm chromatin fiber. (G) An E56R mutant of histone H2A renders IE1-CTD ineffective in altering the structure of the 30-nm chromatin fiber. (H) LANA-CTD (red dots) does not affect the folding of the 30-nm chromatin fiber. Instead, the 10-nm nucleosome array appears to be slightly affected with the addition of LANA-CTD peptide. DOI: http://dx.doi.org/10.7554/eLife.11911.008

Figure 4—figure supplement 1.. Assessment of the quality of reconstituted nucleosomal array.

(A) Nucleosomal arrays corresponding to ~1 μg DNA were cleaved with indicated amount of micrococcal nuclease (MNase). (B) EM analysis of the reconstituted nucleosomal array (Bar: 100 nm). DOI: http://dx.doi.org/10.7554/eLife.11911.009