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W4297896737 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal. Editor's evaluation Giant dsDNA viruses, with genomes in excess of 1Mb encoding more than one thousand genes, were only recently discovered and their study offers new opportunities to probe life's mechanisms. Little is known how these organisms protect and organize their genomes. This fascinating study reveals a helical protein assembly comprised of oxidoreductase-family proteins, which assemble into multi-start helical fibers, with genomic DNA lining the lumen of the fiber. https://doi.org/10.7554/eLife.77607.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Acanthamoeba infecting giant viruses were discovered with the isolation of mimivirus (La Scola et al., 2003; Raoult et al., 2004). Giant viruses now represent a highly diverse group of dsDNA viruses infecting unicellular eukaryotes (Abergel et al., 2015) which play important roles in the environment (Schulz et al., 2020; Moniruzzaman et al., 2020; Kaneko et al., 2021). They also challenge the canonical definitions of viruses (Forterre, 2010; Claverie and Abergel, 2010) as they can encode central translation components (Raoult et al., 2004; Abergel et al., 2007) as well as a complete glycosylation machinery (Piacente et al., 2017; Notaro et al., 2021) among other unique features. Mimivirus has been the most extensively studied giant virus infecting Acanthamoeba (Colson et al., 2017). The virions are 0.75 µm wide and consist of icosahedral capsids of 0.45 µm diameter surrounded by a dense layer of radially arranged fibrils (Raoult et al., 2004). Structural analyses of the virions have provided some insights into the capsid structure (Kuznetsov et al., 2013; Xiao et al., 2009; Klose et al., 2010; Ekeberg et al., 2015; Schrad et al., 2020) but given the size of the icosahedral particles (and hence the sample thickness), accessing the internal organization of the core of the virions remains challenging. Consequently, little is known about the packaging of the 1.2 Mb dsDNA genome (Chelikani et al., 2014). Inside the capsids, a lipid membrane delineates an internal compartment (~340 nm in diameter, Figure 1A, Figure 1—figure supplement 1), referred to as the nucleoid, which contains the viral genome, together with all proteins necessary to initiate the replicative cycle within the host cytoplasm (Schrad et al., 2020; Kuznetsov et al., 2010; Claverie et al., 2009; Arslan et al., 2011). Acanthamoeba cells engulf mimivirus particles, fooled by their bacteria-like size and the heavily glycosylated decorating fibrils (La Scola et al., 2003; Raoult et al., 2004; Notaro et al., 2021). Once in the phagosome, the Stargate portal located at one specific vertex of the icosahedron opens up (Zauberman et al., 2008), enabling the viral membrane to fuse with that of the host vacuole to deliver the nucleoid into the host cytoplasm (Schrad et al., 2020; Claverie et al., 2009). EM studies have shown that next the nucleoid gradually loses its electron dense appearance, transcription begins and the early viral factory is formed (Arslan et al., 2011; Suzan-Monti et al., 2007; Mutsafi et al., 2014). Previous atomic force microscopy studies of the mimivirus infectious cycle suggested that the DNA forms a highly condensed nucleoprotein complex enclosed within the nucleoid (Kuznetsov et al., 2013). Here, we show that opening of the large icosahedral capsid in vitro led to the release of rod-shaped structures of about 30-nm width. These structures were further purified and the various conformations characterized using cryo-electron microscopy (cryo-EM), tomography, and mass spectrometry (MS)-based proteomics. Figure 1 with 10 supplements see all Download asset Open asset The mimivirus genomic fiber. (A) Micrograph of an ultrathin section of resin-embedded infected cells showing the DNA tightly packed inside mimivirus capsids (C) with electron dense material inside the nucleoid (N). The string-like features, most likely enhanced by the dehydration caused by the fixation and embedding protocol, correspond to the genomic fiber (F) packed into the nucleoid. (B) Micrograph of negative stained mimivirus capsid (C) opened in vitro with the genomic fiber (F) still being encased into the membrane-limited nucleoid (N). (C) Multiple strands of the flexible genomic fiber (F) are released from the capsid (C) upon proteolytic treatment. (D) Micrograph of negative stained purified mimivirus genomic fibers showing two conformations the right fiber resembling the one in (B) and free DNA strands (white arrowheads). (E) Slices through two electron cryo tomograms of the isolated helical protein shell of the purified genomic fibers in compact (top) or relaxed conformation (bottom) in the process of losing one protein strand (Figure 1—figure supplement 2, Figure 1—videos 1, 4). (F) Different slices through the two tomograms shown in (E) reveal DNA strands lining the helical protein shell of the purified genomic fibers in compact (top) or relaxed conformation (bottom). Examples of DNA strands extending out at the breaking points of the genomic fiber are marked by black arrowheads. Note in the top panel, individual DNA strands coated by proteins (red arc). The slicing planes at which the mimivirus genomic fibers were viewed are indicated on diagrams on the top right corner as blue dashed lines and the internal colored segments correspond to DNA strands lining the protein shell. The thickness of the tomographic slices is 1.1 nm and the distance between tomographic slices in panels (E) and (F) is 4.4 nm. Scale bars as indicated. Results Capsid opening induces the release of a ~30-nm-wide rod-shaped structure that contains the dsDNA genome We developed an in vitro protocol for particle opening that led to the release of ~30 nm-wide rod-shaped fibers of several microns in length (Figure 1, Figure 1—figure supplement 1). We coined this structure the mimivirus genomic fiber. Complete expulsion of the opened nucleoid content produced bundled fibers resembling a ‘ball of yarn’ (Kuznetsov et al., 2013; Figure 1C). The capsid opening procedure involves limited proteolysis and avoids harsh conditions, as we found that the structure becomes completely denatured by heat (95°C) and is also sensitive to acidic treatment, thus preventing its detection in such conditions (Schrad et al., 2020). Various conformations of the genomic fiber were observed, sometimes even on the same fiber (Figure 1D), ranging from the most compact rod-shaped structures (Figure 1D [left], and Figure 1E, F [top]) to more relaxed structures where DNA strands begin to dissociate (Figure 1D [right], and Figure 1E, F [bottom]). After optimizing the in vitro extraction on different strains of group-A mimiviruses, we focused on an isolate from La Réunion Island (mimivirus reunion), as more capsids were opened by our protocol, leading to higher yields of genomic fibers that were subsequently purified on sucrose gradient. All opened capsids released genomic fibers (Figure 1C, Figure 1—figure supplement 1). The first confirmation of the presence of DNA in the genomic fiber was obtained by agarose gel electrophoresis (Figure 1—figure supplement 3). Cryo-EM bubblegram analysis (Wu et al., 2012; Cheng et al., 2014) gave a further indication that the nucleic acid is located in the fiber lumen. Alike other nucleoprotein complexes, fibers are expected to be more susceptible to radiation damage then pure proteinaceous structures. Surprisingly, the specimen could sustain higher electron irradiation before the appearance of bubbles compared to other studies (Mishyna et al., 2017): 600 e−/Å² for relaxed helices and up to 900 e−/Å² for long compact ones, while no bubbles could be detected in unfolded ribbons (Figure 1—figure supplement 4). For comparison, bacteriophage capsids containing free DNA, that is not in the form of nucleoproteins, show bubbling for doses of ~30–40 e−/Å² (Mishyna et al., 2017). Cryo-EM single-particle analysis of the different compaction states of the mimivirus genomic fiber In order to shed light on the mimivirus genome packaging strategy and to determine the structure of the purified genomic fibers, we performed cryo-EM single-particle analysis. The different conformations of the genomic fiber initially observed by negative staining (Figure 1) and cryo-EM resulted in a highly heterogeneous dataset for single-particle analysis. In order to separate different conformations, in silico sorting through 2D classification (using Relion, He and Scheres, 2017; Scheres, 2012) was performed. Next, we carried out cluster analysis relying on the widths of the helical segments and correlations (real and reciprocal spaces) between the experimental 2D-class patterns (Figure 1—figure supplements 5 and 6 ). Three independent clusters (Cl) could be distinguished, corresponding to the compact (Cl1), intermediate (Cl2), and relaxed (Cl3) fiber conformations (Figure 1—figure supplement 6) with the latter being the widest. For each cluster, we determined their helical symmetry parameters by image power spectra analyses and performed structure determination and refinement (Figure 2, Figure 2—figure supplements 1–3). Figure 2 with 5 supplements see all Download asset Open asset Structures of the mimivirus genomic fiber for Cl1a (A–C), Cl3a (D–F), and Cl2 (G–I): Electron microscopy (EM) maps of Cl1a (A) and Cl3a (D) are shown with each monomer of one GMC-oxidoreductase dimer colored in green and orange and three adjacent dimers in yellow, to indicate the large conformational change taking place between the two fiber states. The transition from Cl1a to Cl3a (5-start helix) corresponds to a rotation of each individual unit (corresponding to a GMC-oxidoreductase dimer) by ~−10° relative to the fiber longitudinal axis and a change in the steepness of the helical rise by ~−11°. Scale bars, 50 Å. Compared to Cl1a, the Cl2 6-start helix (G) shows a difference of ~2° relative to the fiber longitudinal axis and ~2° in the steepness of the helical rise. Scale bars, 50 Å. Cross-sectional (bottom) and longitudinal (top) sections through the middle of final Cl1a (B), Cl3a (E), and Cl2 (H) EM maps. Scale bars, 50 Å. Longitudinal (top) and orthogonal (bottom) views of final Cl1a (C), Cl3a (F), and Cl2 (I) EM maps color coded according to each start of the 5-start helix. Densities for some asymmetric units in the front have been removed on the side view map to show the five DNA strands lining the protein shell interior. Scale bars 50 Å. Figure 3 with 4 supplements see all Download asset Open asset Maps of the compact (Cl1a and Cl2) genomic fiber structures. (A) Cl1a EM map corresponding to the protein shell prior focused refinement is shown as a transparent surface and the five DNA strands as solid surface. One protein dimer strand is shown yellow except for one asymmetric unit (transparent yellow) to illustrate the dimer fit. The position of a second dimer (green) from the adjacent dimer strand is shown to emphasize that the DNA strand (gold) is lining the interface between two dimers. (B) Cartoon representation of GMC-oxidoreductase qu_946 dimers fitted into one of the Cl1a 5-start helix strands in the 3.7-Å resolution map. The map is shown at a threshold highlighting the periodicity of contacts between the dsDNA and the protein shell. Charged distribution on surface representation of Cl1a protein shell made of qu_946 dimers (C) or qu_143 dimers (D). Cartoon representation of qu_946 (E) and qu_143 (F) fitted into the Cl1a cryo-electron microscopy (cryo-EM) maps highlighting the interacting residues (given as stick models) between each monomer and one dsDNA strand. The isosurface threshold chosen allows visualization of density for the manually built N-terminal residues, including terminal cysteines (stick model), of two neighboring monomers that could form a terminal disulfide bridge. (G) Zoom into the 3.3 Å resolution focused refined Cl1a map illustrating the fit of the side chains and the FAD ligand (Figure 3—video 1). (H) Cartoon representation of the DNA fitted in the focused refined DNA only map (Figure 3—figure supplement 2). (I) Focused refined Cl1a map colored by monomer, next to a cartoon representation of the qu_946 dimer (α-helices in red, β-strands in blue, and coils in yellow). Secondary structure elements are annotated in both representations (H: helix, B: beta-strand). For both Cl1a and Cl3a conformations, after 3D refinement, we obtained helical structures of 3.7 Å resolution (Fourrier shell correlation [FSC] threshold 0.5, masked), corresponding to 5-start left-handed helices made of a ~8-nm-thick proteinaceous external shell (Figure 2—figure supplements 1–2). For the most compact conformation (Cl1a) five dsDNA strands were lining the interior of the protein shell leaving a ~9-nm-wide central channel (Figures 2 and 3, Supplementary file 1). The dsDNA strands appear as curved cylinders in the helical structure, the characteristic shape of the DNA (minor and major groove) becoming only visible after focused refinement of a single strand of dsDNA (Figure 3, Figure 3—figure supplements 1 and 2). In the relaxed subcluster Cl3a, the DNA strands at the interface to the ~17-nm-wide central channel are not clearly recognizable (Figure 2, Supplementary file 1, and Figure 2—figure supplement 2), most likely because they are at least partially detached inside the broken expanded fiber. The breaks after relaxation of the helix might be the result of the extraction and purification treatment, while DNA will remain in the central channel, at least in the early phase of Acanthamoeba infection. Finally, the 4-Å resolution Cl2 map obtained after 3D refinement (Figure 2—figure supplement 3) corresponds to a 6-start left-handed helix made of a~8-nm-thick proteinaceous external shell, with six dsDNA strands lining the shell interior and leaving a ~12-nm-wide inner channel (Figure 2, Supplementary file 1). The most abundant proteins in the genomic fiber are GMC oxidoreductases, the same that compose the fibrils decorating mimivirus capsid MS-based proteomic analyses performed on three biological replicates identified two GMC oxidoreductases as the main components of the purified genomic fiber (qu_946 and qu_143 in mimivirus reunion corresponding to L894/93 and R135, respectively, in mimivirus prototype) (Supplementary file 2). The two mimivirus reunion proteins share 69% identity (81% similarity). The available mimivirus R135 GMC-oxidoreductase dimeric structure (Klose et al., 2015) (PDB 4Z24, lacking the 50 amino acid long cysteine-rich N-terminal domain) was fitted into the EM maps (Figures 2 and 3). This is quite unexpected, since GMC oxidoreductases are already known to compose the fibrils surrounding mimivirus capsids (Notaro et al., 2021; Boyer et al., 2011). The corresponding genes are highly expressed during the late phase of the infection cycle at the time of virion assembly. Notably, the proteomic analyses provided different sequence coverages for the GMC oxidoreductases depending on whether samples were intact virions or purified genomic fiber preparations, with substantial under-representation of the N-terminal domain in the genomic fiber (Figure 2—figure supplement 4). Accordingly, the maturation of the GMC oxidoreductases involved in genome packaging must be mediated by one of the many proteases encoded by the virus or the host cell. Interestingly, mimivirus M4 (Boyer et al., 2011), a laboratory strain having lost the genes responsible for the synthesis of the two polysaccharides decorating mimivirus fibrils (Notaro et al., 2021) also lacks the GMC-oxidoreductase genes. Additional studies on this specific variant will be key to establish if it exhibits a similar genomic fiber, and if yes, which proteins are composing it. Analysis of the genomic fiber structure The EM maps and FSC curves of Cl1a are shown in Figure 2—figure supplement 1. An additional step of refinement focused on the asymmetric unit further improved the local resolution to 3.3 Å as indicated by the corresponding FSC (Figure 3 and Figure 2—figure supplement 1). After fitting the most abundant GMC-oxidoreductase qu_946 (SWISS-MODEL model Waterhouse et al., 2018) in the final map of Cl1a, five additional N-terminal residues in each monomer were manually built using the uninterpreted density available. This strikingly brings the cysteines of each monomer (C51 in qu_946) close enough to allow a disulfide bridge, directly after the 50 amino acids domain not covered in the proteomic analysis of the genomic fiber (Figure 3G, Figure 2—figure supplement 4). The N-terminal chain, being more disordered than the rest of the structure, it is absent in the focused refined map, and also absent in the Cl3a map of the relaxed helix, suggesting that a break of the disulfide bridge could be involved in the observed unwinding process. Models of the three helical assemblies and asymmetric unit were further refined using the real-space refinement program in PHENIX 1.18.2 (Liebschner et al., 2019). In the 3.3-Å resolution map of the asymmetric unit, most side chains and notably the FAD cofactor are accommodated by density suggesting that the oxidoreductase enzyme could be active (Figure 3D). Density that can be attributed to the FAD cofactor is also present in the Cl2 and Cl3a maps. The atomic models of Cl1a and Cl3a dimers are superimposable with a core root-mean-square deviation (RMSD) of 0.68 Å based on Cα atoms. Inspection of individual genomic fibers in the tomograms confirmed the coexistence of both 5- and 6-start left-handed helices containing DNA (Figure 1—figure supplement 2 and Figure 3—animation 1, Figure 1—video 1). Further, some intermediate and relaxed structures were also observed in which the DNA segments appeared detached from the protein shell and sometimes completely absent from the central channel of the broken fibers. Both GMC oxidoreductases (qu_946 and qu_143) can be fitted in the 5- and 6-start maps. In relaxed or broken fibers, large electron dense structures that might correspond to proteins inside the lumen were sometimes visible (Figure 1—figure supplement 2C, E and Figure 1—video 3) as well as dissociating DNA fragments, either in the central channel (Figure 1—figure supplement 2B and Figure 1—video 2) or at the breakage points of the fibers in its periphery (Figure 1—figure supplement 2D and Figure 1—video 4). Densities corresponding to the dimer subunits composing the protein shell were also commonly observed on dissociated DNA strands (Figure 1E, F, Figure 1—figure supplement 2, and Figure 1—video 1; Figure 1—video 2; Figure 1—video 3; Figure 1—video 4). In the Cl1a and Cl2 helices, the monomers in each dimer are interacting with two different dsDNA strands. As a result, the DNA strands are interspersed between two dimers, each also corresponding to a different strand of the protein shell helix (Figure 3). Based on the periodic contacts between protein shell and DNA strands, these interactions might involve, in the case of the Cl1a helix, one aspartate (D82 relative to the N-terminal methionine in qu_946), one glutamate (E321), two lysines (K344, K685), one arginine (R324), and a histidine (H343) or one asparagine (N80), two lysines (K319, K342), one arginine (R322), and one tyrosine (Y687), in the case of the qu_143 (Figure 3E, F, Supplementary file 3). Intra- and interstrands contacts between each dimer are presented in Supplementary file 3 for qu_143 and qu_946 in Cl1a, Cl2, and Cl3 maps. Despite the conformational heterogeneity and the flexibility of the rod-shaped structure, we were able to build three atomic models of the mimivirus genomic fiber, in compact (5- and 6-start) and relaxed (5-start) states. Higher resolution data would still be needed to determine the precise structure of the dsDNA corresponding to the viral genome (Figure 3B, H), however, the lower resolution for this part of the map even in focused refinement runs (Figure 3H) might also mean that the DNA does not always bind in the same orientation. Rough estimation of genome compaction to fit into the nucleoid Since there is a mixture of five and six strands of DNA in the genomic fiber, this could correspond to five or six genomes per fiber or to a single folded genome. Assuming that the length of DNA in B-form is ~34 Å for 10 bp, the mimivirus linear genome of 1.2 × 106 bp would extend over ~400 µm and occupy a volume of 1.3 × 106 nm3 (~300 µm and ~1 × 106 nm3 if in A-form) (Li et al., 2019). The volume of the nucleoid (Kuznetsov et al., 2010) (~340 nm in diameter) is approximately 2.1 × 107 nm3 and could accommodate over 12 copies of viral genomes in a naked state, but only 40 µm of the ~30-nm-wide flexible genomic fiber. Obviously, the mimivirus genome cannot be simply arranged linearly in the genomic fiber and must undergo further compaction to accommodate the 1.2 Mb genome in a ~40-µm-long genomic fiber. As a result, the complete mimivirus genome, folded at least five times, fits into the helical shell. This structure surprisingly resembles a nucleocapsid, such as the archaea infecting APBV1 nucleocapsid (Ptchelkine et al., 2017). Additional proteins, including RNA polymerase subunits, are enriched in the genomic fiber The proteomic analysis of fiber preparations revealed the presence of additional proteins including several RNA polymerase subunits: Rpb1 and Rpb2 (qu_530/532 and qu_261/259/257/255), Rpb3/11 (qu_493), Rpb5 (qu_245), RpbN (qu_379), and Rpb9 (qu_219), in addition to a kinesin (qu_313), a regulator of chromosome condensation (qu_366), a helicase (qu_572), to be possibly associated with the genome (Supplementary file 2). In addition to the two GMC oxidoreductases, at least three oxidative stress proteins were also identified together with hypothetical proteins (Supplementary file 2). RNA polymerase subunits start being expressed 1hr postinfection with a peak after 5 hr and are expressed until the end the infection cycle. GMC oxidoreductases, kinesin, regulator of chromosome condensation are all expressed after 5 hr of infection until the end of the cycle. As expected, the core protein (qu_431) composing the nucleoid and the major capsid proteins (MCP, qu_446) were significantly decreased in the genomic fiber proteome compared to intact virions. In fact, qu_431 and qu_446 represent, respectively, 4.5% and 9.4% of the total protein abundance in virions whereas they only account for 0.4% and 0.7% of the total protein abundance in the genomic fiber, suggesting that they could be contaminants in this preparation. On the contrary, we calculated enrichment factors of more than 500 (qu_946) and 26 (qu_143) in the genomic fiber samples compared to the intact virion. Finally, the most abundant RNA polymerase subunit (qu_245) is increased by a factor of eight in the genomic fiber compared to intact virion (if the six different subunits identified are used, the global enrichment is sevenfold). Furthermore, upon inspection of the negative staining micrographs, macromolecules strikingly resembling the characteristic structure of the poxviruses RNA polymerase (Grimm et al., 2019) were frequently observed scattered around the unwinding fiber and sometimes sitting on DNA strands near broken fibers (Figure 4). Together with the tomograms showing large electron dense structures in the lumen, some RNA polymerase units could occupy the center of the genomic fiber. Figure 4 Download asset Open asset RNA polymerase could be associated to the genomic fiber. (A) Micrograph of negative stained fiber with released DNA still being connected to a relaxed and broken fiber and adjacent scattered macromolecular complexes that might resemble RNA polymerases. (B) Strikingly, some of them (black arrows) appear to sit on a DNA strand (white arrow). (C) E, particle extracted from the NS-TEM image; P, projections of vaccinia virus RNA polymerase (6RIC and 6RUI, Grimm et al., 2019) structure in preferred orientation; CE, clean extraction (see Material and methods). White and black boxed corresponds to images with white and black asterisk (CE), respectively. Scale bar, 50 Å. Negative staining imaging may dehydrate the objects and change macromolecules volumes. Discussion Several DNA compaction solutions have been described. For instance, the DNA of filamentous viruses infecting archaea is wrapped by proteins to form a ribbon which in turn folds into a helical rod forming a cavity in its lumen (DiMaio et al., 2015; Wang et al., 2020). In contrast, the chromatin of cellular eukaryotes consists of DNA wrapped around histone complexes (Robinson et al., 2006). It was recently shown that the virally encoded histone doublets of the Marseilleviridae can form nucleosomes (Liu et al., 2021; Valencia-Sánchez et al., 2021) and such organization would be consistent with previous evolutionary hypotheses linking giant DNA viruses with the emergence of the eukaryotic nucleus (Bell, 2001; Bell, 2020; Chaikeeratisak et al., 2017; Claverie, 2006; Takemura, 2001). Herpesviruses (Gong et al., 2019; Liu et al., 2019), bacteriophages (Sun et al., 2015; Rao and Feiss, 2015), and APBV1 archaeal virus (Ptchelkine et al., 2017) package their dsDNA genome as naked helices or coils. Yet, APBV1 nucleocapsid structure strikingly resemble the mimivirus genomic fiber with a proteinacious shell enclosing the folded dsDNA genome. Consequently, mimivirus genomic fiber is a nucleocapsid further bundled as a ball of yarn into the nucleoid, itself encased in the large icosahedral capsids. The structure of the mimivirus genomic fiber described herein supports a complex assembly process where the DNA must be folded into five or six strands prior to or concomitant with packaging, a step that may involve the repeat containing regulator of chromosome condensation (qu_366) identified in the proteomic analysis of the genomic fiber. The proteinaceous shell, via contacting residues between the dsDNA and the GMC oxidoreductases, would guide the folding of the dsDNA strands into the structure prior loading into the nucleoid. The lumen of the fiber being large enough to accommodate the mimivirus RNA polymerase, we hypothesize that it could be sitting on the highly conserved promoter sequence of early genes (Suhre et al., 2005). This central position would support the involvement of the RNA polymerase in genome packaging into the nucleoid and could determine the channel width via its anchoring on the genome (Figure 4D). According to this scenario, the available space (although tight) for the RNA polymerase inside the genomic fiber lumen suggests it could be sterically locked inside the compact form of the genomic fiber and could start moving and transcribing upon helix relaxation, initiating the replicative cycle and the establishment of the cytoplasmic viral factory. The genome and the transcription machinery would thus be compacted together into a proteinaceous shield, ready for transcription upon relaxation (Figure 2—video 1). This organization would represent a remarkable evolutionary strategy for packaging and protecting the viral genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. This is conceptually reminiscent of icosahedral and filamentous dsRNA viruses which pack and protect their genomes together with the replicative RNA polymerase into an inner core (Toriyama, 1986; Collier et al., 2016; Ding et al., 2019). As a result, replication and transcription take place within the protein shield and viral genomes remain protected during their entire infectious cycle. In the case of dsDNA viruses however, the double helix must additionally open up to allow transcription to proceed, possibly involving the helicase identified in our proteomic study (Supplementary file 2). Finally, in addition to their structural roles, the FAD containing GMC oxidoreductases making the proteinaceous shield, together with other oxidative stress proteins (Supplementary file 2), could alleviate the oxidative stress to which the virions are exposed while entering the cell by phagocytosis. Mimivirus virion thus appears as a Russian doll, with its icosahedral capsids covered with heavily glycosylated fibrils, two internal membranes, one lining the capsid shell, the other encasing the nucleoid, in which the genomic fiber is finally folded. To our knowledge, the structure of the genomic fiber used by mimivirus to package and protect its genome in the nucleoid r" @default.
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W4297896737 title "Decision letter: The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield" @default.
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