doi: 10.1016/j.cell.2021.06.032.
Epub 2021 Jul 22.
Virus-encoded histone doublets are essential and form nucleosome-like structures
Affiliations
- PMID: 34297924
- PMCID: PMC8357426
- DOI: 10.1016/j.cell.2021.06.032
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Virus-encoded histone doublets are essential and form nucleosome-like structures
Cell.
.
Abstract
The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of "minimalist" histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of "organisms" that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.
Keywords: Analytical Ultracentrifugation; KO fitness impact; Melbournevirus genetics; NCLDV; acidic patch; cryo-EM; doublet histone; giant virus; histone tail; non-eukaryotic nucleosome; nucleosome-like-particle; viral factory; viral nucleosome.
Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
Declaration of interests The authors declare no competing interests.
Figures
MV histone doublets are re-localized to viral factories (VFs) (A) Histone dimer pairs (H2B-H2A and H4-H3) within Eukarya were aligned against the doublet Marseilleviridae histones using HHpred’s multiple sequence alignment tool (ClustalΩ). Known α helices from the histone fold (HF) domain in Eukarya are dark-colored tubes (H2B, red; H2A, yellow; H4, green; H3, blue; and additional helices, gray). Predicted α helices in MV histones were generated using HHrped’s Quick 2D prediction web server (shown in lighter coloration) within the Marseilleviridae histone doublets. Known R-T pairs and DNA binding residues are shown in Eukarya histones along with their conservation within Marseilleviridae histones; additional predicted DNA-binding residues are shown (positions demonstrated by lollipops). (B) Light microscopy fluorescence images (scale bar, 10 μm) of A. castellanii cells transfected with GFP-A. castellanii-H2A, MV-H2B-H2A-GFP, and MV-H3-H4-GFP, non-infected and infected with MV at 4 h PI. While GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells, MV-H2B-H2A-GFP and MV-H3-H4-GFP are scattered in the entire cell (including the nucleus). Upon virus infection, GFP-A. castellanii-H2A remains in the nucleus (yellow arrows), while MV-H2B-H2A-GFP and MV-H3-H4-GFP re-localize to the VF (cyan arrows). DAPI staining remains in the nucleus all along the infection, but the intense fluorescence in the late VF hides the staining of the nucleus at 4 h PI. See also Figure S1.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
Localization of fluorescently labeled MV histones in virus-infected Amoeba (A) Complete sequence alignment of H2B-H2A (top) and H4-H3 (bottom) from Marseilleviridae, the host Acanthamoeba castellanii, and Xenopus laevis. Predicted α helices of H2B-H2A (light red and yellow) and H4-H3 (light green and blue) Melbournevirus histone doublets were generated using HHPRED’s Quick 2D prediction web server. Histone dimer pairs H2B-H2A and H4-H3 of Acanthamoeba castellanii and Xenopus laevis were each aligned against their respective Marseilleviridae histone doublets using HHPRED’s multiple sequence alignment tool, ClustalΩ. Conservation of each specific residue in each alignment is denoted by blue shading, with greater conservation being represented by darker blue. Known R-D clamp, R-T pairs, and DNA binding residues are indicated for Xenopus laevis histone pairs with their conservation within Melbournevirus histones. Marseilleviridae histones are 16%–33% conserved to Xenopus laevis histones (shown on far right of alignment). Known α helices from the histone fold domain in Xenopus laevis are shown as dark colored tubes; H2B are red, H2A are yellow, H4 are green, H3 in blue, and additional helices in gray. Logo plot demonstrating residue conservation among the alignments provided by ClustalΩ tool is shown below. Light microscopy fluorescence images (scale bar 10 μm) of A. castellanii cells transfected with (B) GFP-A. castellanii-H2A, C) MV-H2B-H2A-GFP, D) MV-H4-H3-GFP and E) MV-miniH2B-H2A-GFP, non-infected (NI) and infected with Melbournevirus at 30 min, 1h, 2h, 3h and 4h pi. B) GFP-A. castellanii-H2A concentrates only in the nucleus (N) of the non-infected cells and all along the infectious cycle. (C) MV-H2B-H2A-GFP, (D) MV-H4-H3-GFP and (E) MV-miniH2B-H2A-GFP are scattered in the entire cell (including the nucleus) in the non-infected cells. Between 1h and 2h pi, the viral histones start accumulating in the early viral factories (eVF). At 4h pi, the fluorescence is predominantly concentrated in the mature viral factory (VF). DAPI staining remains at the nucleus all along the infection but the intense fluorescence in the late VF hides the staining of the nucleus at 4h pi. (F). MV H2B-H2A-GFP and MV H4-H3-mRFP are co-localized in the viral factory, along with the viral DNA at 4h pi. (G). DAPI staining of Acanthamoeba cells infected with Melbournevirus at 4h pi. The host nucleus does not disappear upon the infectious cycle. Due to the large accumulation of viral DNA in the viral factories, exposure time for the DAPI fluorescence was optimized to visualize both the viral factory (VF) and the nucleus (N) of the cell (typically between 400ms and 1 s). (H) Schematic depicting the strategy for analysis of histone gene knockouts. Related to Figures 1 and 2.
MV histone doublets are essential for virus fitness (A) Histone (MV and host) expression and host histone chaperones expression profile during the MV infectious cycle. Host actin genes expression were used for reference. (B) Schematic representation of the strategy used to generate the recombinant mel_368 or mel_369 KO. Expected PCR product size are also shown. Primer a: HB146 or HB147 and primer b: HB145 or HB148 for mel_368 and mel_369 respectively; primer c, GFP reverse; primer d, GFP forward (Table S1). goi, gene of interest. (C) PCR demonstrates correct integration of vectors in the locus of mel_368 or mel_369. Analysis of fitness changes associated with histone KO was analyzed by virus competition assay in wild-type or complemented amoebas. Mock cells were incubated with Superfect in the absence of plasmid. a+c, 5′ integration; d+b, 3′ integration; a+b, wild-type locus. HR, homologous recombination. See also Figure S1 and Table S1.
MV-histones form nucleosome-like particles (A) SDS-PAGE of purified Melbournevirus (MV) histone doublets. (B) Sucrose gradient sedimentation of MV NLPs with 147 or 207 bp DNA. The compositions of MV NLPs were analyzed by native- and SDS-PAGE. (C) MV-NLP and eNuc were reconstituted on Widom ‘601’ DNA and Melbournevirus native DNA, respectively. The MV-NLP and eNuc were heat treated at 37 and 55°C. (D) Native PAGE of reconstituted MV mini-NLP (mini H2B-H2A instead of H2B-H2A) with 147 bp DNA. (E) GraFix of MV-NLPs with 207 bp DNA (top two panels) and 147 bp DNA (bottom two panels. Native- and SDS- PAGE of the crosslinked MV-NLP fractions representing successful crosslinking, compared to the native MV-NLP input. (F) Representative AFM images: Samples were diluted in TCS buffer and applied to APTES coated mica, rinsed with water and imaged on a NanoWizard Sa with a TAP300-GD cantilever. Samples include 147 bp DNA only, eNuc147, MV-H4-H3 with 147 bp DNA, MV-NLP147, MV-NLP207 and MV-NLP207 GraFix. Related to Figure 3.
MV-histones form nucleosome-like particles (A) SDS-PAGE of purified Melbournevirus (MV) histone doublets. (B) Sucrose gradient sedimentation of MV NLPs with 147 or 207 bp DNA. The compositions of MV NLPs were analyzed by native- and SDS-PAGE. (C) MV-NLP and eNuc were reconstituted on Widom ‘601’ DNA and Melbournevirus native DNA, respectively. The MV-NLP and eNuc were heat treated at 37 and 55°C. (D) Native PAGE of reconstituted MV mini-NLP (mini H2B-H2A instead of H2B-H2A) with 147 bp DNA. (E) GraFix of MV-NLPs with 207 bp DNA (top two panels) and 147 bp DNA (bottom two panels. Native- and SDS- PAGE of the crosslinked MV-NLP fractions representing successful crosslinking, compared to the native MV-NLP input. (F) Representative AFM images: Samples were diluted in TCS buffer and applied to APTES coated mica, rinsed with water and imaged on a NanoWizard Sa with a TAP300-GD cantilever. Samples include 147 bp DNA only, eNuc147, MV-H4-H3 with 147 bp DNA, MV-NLP147, MV-NLP207 and MV-NLP207 GraFix. Related to Figure 3.
Histone doublets in Marseilleviridae form nucleosome-like particles (MV-NLPs) (A) Native PAGE of reconstituted MV-NLPs with “601” DNA of various lengths. Left panel: individual and combinations of histone doublets reconstituted onto 147 bp DNA; right panel: MV-H2B-H2B and MV-H4-H3 reconstituted onto 601 DNA of varying lengths. (B) Sedimentation velocity analytical ultracentrifugation (SV-AUC) of MV-NLPs. Left: van Holde-Weischet plot of eukaryotic nucleosomes (eNuc), histone-DNA complexes with individual MV histone doublets, and native MV-NLPs with 147 bp DNA (no GraFix); right: van Holde-Weischet plot of crosslinked (GraFix-ed) MV-NLPs with 123, 147, or 207 bp DNA. (C) Height profile of MV-NLPs with 147 or 207 bp DNA, obtained by atomic force microscopy (AFM). The average height profiles with standard deviations of the particles from each sample are shown; representative, original images are shown in Figure S2, and statistics are shown in Table S2.
Cryo-EM data analysis of MV-NLP207 and MV-NLP147 (A) Raw micrograph of the Grafix-treated MV-NLP207 (Scale bar is 50 nm), 2D class averages generated from the dataset, 3D structure of the MV NLP, local resolution map and FSC curve, and data processing strategy flow chart. (B) Raw micrograph of the GraFix treated MV-NLP147 (Scale bar is 50 nm), 2D class averages generated from the dataset, 3D structure of the MV NLP, local resolution map and FSC curve, and data processing strategy flow chart. Related to Figure 4.
Cryo-EM data analysis of MV-NLP207 and MV-NLP147 (A) Raw micrograph of the Grafix-treated MV-NLP207 (Scale bar is 50 nm), 2D class averages generated from the dataset, 3D structure of the MV NLP, local resolution map and FSC curve, and data processing strategy flow chart. (B) Raw micrograph of the GraFix treated MV-NLP147 (Scale bar is 50 nm), 2D class averages generated from the dataset, 3D structure of the MV NLP, local resolution map and FSC curve, and data processing strategy flow chart. Related to Figure 4.
Cryo-EM reveals that MV-histone doublets form nucleosome-like structures with asymmetrically extending DNA (A) Overview of MV-NLP147 and electron density. The equivalent regions of MV H3, H4, H2A, and H2B are shown in blue, green, yellow, and red, respectively. (B) Overlay of MV-NLP147 (blue) with eNuc (gray). Only 80 bp of DNA with associated histones are shown for clarity. Superhelix locations (SHLs) are numbered from 0 to 6 starting from the nucleosome dyad (φ). (C) Comparison of the DNA path of eNuc147 (gray ribbon diagram), MV-NLP147 (blue electron density), and MV-NLP207 (green electron density). (D) Charged surface representation of the histones for MV-NLP147 and eNuc147. Coordinates for eNuc147 were taken from 3LZ0. See also Figures S3A and S3B and Table S3.
Comparison of MV-NLP and eNuc histone structures (A) Superposition of two MV-H4-H3 doublets (green and blue) with the eukaryotic (H3-H4)2 tetramer in gray (left), and a close-up of MV-H4-H3 doublet connector (right). Interactions of the MV-H4 N-terminal tail with the nucleosome are also shown. (B) A comparison of the four-helix bundle structure formed by H4 and H2B. (C) Superposition of two MV-H2B-H2A doublets (in red and yellow) with eukaryotic H2A-H2B dimers in gray (left), and a close-up of the MV-H2B-H2A doublet connector and docking domain. See Table S4 for detailed information. Additional possible configurations for both connectors are shown in Figure S4.
MV-nucleosome histone dimer arrangement and histone connectors (A) Description of ϕ, the pseudo-dihedral angle measuring the relative orientations of the H2B-H2A subunits, between MV-NLP and eNuc. Larger magnitude values correspond to a dimer arrangement that is angled further away from the dyad axis. Fictitious particle locations are shown as black spheres, and histones (H3 in blue, H4 in green, H2A in yellow, and H2B in red) and DNA (silver and gold ribbons) are semi-transparent to allow for better visibility of each particle’s location. (B) Overlay of de novo loop models with feasible conformations connecting H2B to H2A and (C) H4 to H3 in the MV-histone doublets. Loop configurations were generated using Modeler (v9.20) and then placed in the nucleosome context. After manually removing physically non-relevant (knots) conformations and clashes identified with a cutoff distance of 0.8 Å using CPPTRAJ of the Amber MD package (v18), the three best loops are shown in different colors (cross correlation range against the simulated map is 0.614-0.623). Related to Figure 5.
Comment in
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A small nucleosome from a weird virus with a fat genome.Mol Cell. 2021 Sep 2;81(17):3447-3448. doi: 10.1016/j.molcel.2021.08.014. Mol Cell. 2021. PMID: 34478653
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