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Cisternae and vesicles apposing viral factories at the onset of capsid assembly. A. 10 nm digital slice derived from a 280 nm-thick STEM tomogram of a 7.5-hour PI viral factory. The slice reveal cisternae (arrows) and clusters of ~70 nm vesicles (arrowheads) at close vicinity to the viral factory (VF). B. Magnification of the delineated regions in (A). C. A different digital slice from the STEM tomogram shown in (A), with 3-dimensional surface rendering of vesicles; numbers correspond to the regions delineated in (A) and (B). The insets depict magnified 3-dimensional surface rendering of the vesicles from region 2 at two different angles, demonstrating the spherical structure of the vesicles (Movie S3). D. 10 nm digital slice from a different 280 nm-thick STEM tomogram of a 7.5-hour PI viral factory, showing membrane structures surrounded by multiple vesicles near viral factories (Movie S4). The inset demonstrates that membrane structures apposing the factories are studded with ribosomes (black arrows). E, F. Two STEM tomogram slices of an 8-hour PI viral factory that are derived from serial sections and are located 480 nm apart. These tomograms reveal that as Mimivirus progeny are assembled, host cisternae are excluded from the membrane assembly zone (Movies S5, S6). Scale bars: 200 nm in A, D; 100 nm in B; 250 nm in E.
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Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant...
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... of fully assembled icosahedral capsids at the periphery of viral factories at ,8 hours PI. To obtain insights into earlier stages of capsid generation, STEM tomography analyses were conducted on thick (280-320 nm) sections of infected cells at 7.5 hours PI. These analyses revealed membrane cisternae localized at close proximity to the factories ( Fig. 2A-D; Movies S3, S4). Notably, the cisternae do not enwrap the whole factory but rather are detected at discrete sites that consistently coincide with regions where angular structures are detected. This observation, along with the finding that neither cisternae nor angular structures are present in earlier PI times, imply a causal ...
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... PI cisternae are detected at 50 to 100 nm from the edge of the factory core, at 8 hours PI, when partially and fully assembled icosahedral capsids already surround the entire viral factory, these membrane structures are observed at signifi- cantly larger distances (,500 nm) from the factory core, apparently excluded by newly assembling capsids ( Fig. 2E, F; Movies S5, S6). However, multiple ,70 nm vesicles are present near the cisternae as well as within the inner, membrane assembly zone, thus supporting the notion that the vesicles are derived from the cisternae. Notably, this finding implies that these ...
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... vesicles near viral factories (Movie S4). The inset demonstrates that membrane structures apposing the factories are studded with ribosomes (black arrows). E, F. Two STEM tomogram slices of an 8-hour PI viral factory that are derived from serial sections and are located 480 nm apart. These factories are occasionally studded with ribosomes ( Fig. 2D; Movie S4), thus revealing a characteristic appearance of rough endo- plasmic reticulum (RER). The origin of the Mimivirus membranes was also investigated by using antibodies against common endoplasmic reticulum markers. These included protein disulfide isomerase (PDI), a soluble protein residing in the ER lumen, and the KDEL retention ...
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... evaluate this idea we raised antibodies against L425 in both mice and rabbits by using a mixture of three peptides derived from L425 that were estimated as particularly immunogenic. The resulting antibodies recognized a single ,70 kDa band in lysates of both infected host cells and purified viruses (Fig. S2), thus confirming that these antibodies interact with L425, whose calculated weight is 67.27 kDa. Immunolabeling with anti-L425 antibodies of both cryo-preserved ( Fig. 6A-D) and chemically- fixed ( Fig. 6E-G) sections of 8 hours PI cells, but not of any cell sections derived from earlier PI time points, resulted in clear tomograms ...
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... in clear tomograms reveal that as Mimivirus progeny are assembled, host cisternae are excluded from the membrane assembly zone (Movies S5, S6). Scale bars: 200 nm in A, D; 100 nm in B; 250 nm in E. doi:10.1371/journal.ppat.1003367.g002 labeling of angular structures, of assembling icosahedral morphol- ogies as well as of fully assembled capsids (Fig. 6 and Fig. ...
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Citations
... Fewer studies described CPE in A. polyphaga during GVs infection. Acanthamoeba polyphaga mimivirus (APMV) infection leads to nucleus enlargement, prominent VF formation in the cytoplasm, multivesicular bodies and endoplasmic reticulum (ER) membranes appearance around VF, stimulus of ER synthesis, significant structural alteration in microtubules and actin microfilaments resulting in cells becoming smooth, rounded, and losing motility [83][84][85] . Marseillevirus infection induces similar VFs to the observed in A. castellanii, with lack of a clear boundary with cytoplasm 67,86 . ...
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... Vesicular tr affic king is likel y a critical component of virion morphogenesis that recruits membranes derived from the endoplasmic reticulum (ER) to the virus factory, where they ultimatel y giv e rise to the inner membrane of new virions (Mutsafi et al. 2013, Suárez et al. 2013. Treatment with vesicular trafficking inhibitors leads to defective virion morphogenesis in marseilleviruses (Arantes et al. 2016 (Wilson et al. 2005, Legendre et al. 2010. ...
The phylum Nucleocytoviricota includes the largest and most complex viruses known. These “giant viruses” have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host - referred to here as viral homologs or “virologs” - as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery “imitates” immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research.
... Despite not providing clear evidence of an evolutionary relationship between the entire nucleus and the virion factory of Nucleocytoviricota, these latter accumulate intriguing elements that strongly suggest one ( Figure 1). Structurally, the virion factory of giant viruses indeed exhibits notable analogies with the nucleus [61][62][63][64][65][66]: many viruses recruit parts of the endoplasmic reticulum membrane to form their factory, which occasionally assembles close to the microtubule organizing center, akin to nuclear mitosis. In the case of Mimivirus, the virion factory is even formed directly from the fusion of membrane vesicles that likely originate from invaginations of the nuclear membrane [67]. ...
Our perception of viruses has been drastically evolving since the inception of the field of virology over a century ago. In particular, the discovery of giant viruses from the Nucleocytoviricota phylum marked a pivotal moment. Their previously concealed diversity and abundance unearthed an unprecedented complexity in the virus world, a complexity that called for new definitions and concepts. These giant viruses underscore the intricate interactions that unfold over time between viruses and their hosts, and are themselves suspected to have played a significant role as a driving force in the evolution of eukaryotes since the dawn of this cellular domain. Whether they possess exceptional relationships with their hosts or whether they unveil the actual depths of evolutionary connections between viruses and cells otherwise hidden in smaller viruses, the attraction giant viruses exert on the scientific community and beyond continues to grow. Yet, they still hold surprises. Indeed, the recent identification of mirusviruses connects giant viruses to herpesviruses, each belonging to distinct viral realms. This discovery substantially broadens the evolutionary landscape of Nucleocytoviricota. Undoubtedly, the years to come will reveal their share of surprises.
... Observation of individual symmetrons in the samples of Sericesthis Iridescent Virus 103 and Tipula Iridescent Virus 104 support the hypothesis of the proposed symmetron model, in which capsomers are pre-assembled into the trisymmetrons and pentasymmetrons that are then assembled into the icosahedron with the help of the mCPs, such as the zip proteins 34 . However, this model is inconsistent with the continuous assembly process observed in many in situ studies for NCV-infected cells 84,94,[105][106][107] . ...
... This assembly model first illustrated that the fivefold vertex act as the initiation site. Some EM studies for vaccinia virus 105,106 , PBCV-1 84 , and mimivirus 94,107 display some architectures containing the fivefold vertex in the early VF, supporting this notion. Furthermore, these studies also demonstrate that initiating capsid assembly requires capsid precursors to link with precursors of the inner membrane. ...
... Our findings demonstrate that VP88 (P5), which is located close to the edge of the pentasymmetron (Fig. 3d, f) and anchored to the viral inner membrane (Fig. 3g), is associated with the ER (Fig. 5a, d). Combined with previous observations 78,84,[89][90][91][92][93][94][95] , these results indicate that VP88 could function at a very early stage and be pivotal for initiating capsid assembly. Additionally, immunofluorescence assay shows that the SGIV mCPs are complexed with MCP to form various building blocks in the cytoplasm during the early stage of viral infection. ...
Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.
... These phages use scaffold proteins to build a uniform icosahedral capsid and then release the proteins after the assembly of the capsid is complete and before the viral DNA is incorporated into the capsid. In NCLDVs such as mimivirus (20), ASFV (21), vaccinia virus (22), and Paramecium bursaria chlorella virus (PBCV-1) (23), the formation of virus particles begins with a membrane sheet scaffolded with proteins, and the resultant curled The color scheme used to indicate the distribution of the three types of spikes on the hexagon is the same as in panels A and B. Orientations of MCP trimers are indicated using different colors. The central pentasymmetron (salmon pink) and surrounding five trisymmetrons are outlined using colored lines. ...
... In iridovirus (26) and PBCV-1 (23), which form a viral factory near the host nucleus, capsid production occurs in the viral factory, and viral DNA replicated inside the host nucleus is delivered to the viral factory for encapsulation by the capsid. Mimivirus (20) and vaccinia virus (27), which also form a viral factory, produce the capsids around the viral factory and incorporate the viral DNA that is replicated in the viral factory itself. Thus, these NCLDVs package DNA with greater efficiency than medusavirus, in which DNA packaging accidentally occurs near the host nucleus because of the random distribution of empty particles in the cytoplasm (Fig. 3). ...
Giant viruses exhibit diverse morphologies and maturation processes. In this study, medusavirus showed four types of particle morphologies, both inside and outside the infected cells, when propagated in amoeba culture.
... These viruses contain several complex biological systems, such as the mimivirus virophage resistance element (MIMIVIRE) (15) and many genes responsible for protein synthesis, including aminoacyl-tRNA synthetase (aa-RS) genes (1-5). Notably, members of the family Mimiviridae can build a cytoplasmic, organelle-like virion factory (VF) in host cells (16)(17)(18). The VF contains large amounts of replicating mimivirus genomic DNA, and new virions appear on the surface of the VF, which contains materials for capsids and inner membranes. ...
... The VF contains large amounts of replicating mimivirus genomic DNA, and new virions appear on the surface of the VF, which contains materials for capsids and inner membranes. The mimivirus VF is proposed to be localized close to the endoplasmic reticulum (ER) and derive its inner membranes from the ER or from ER-related vesicles (16)(17)(18). ...
... A previous study has shown that membrane assembly of mimivirus particles occurs at the edge of VFs. The inner membrane of mimivirus particles has also been suggested to be derived from the ER (16)(17)(18). However, based on our observation of the structures around early VFs and the colocalization of GM130 to the edge of matured VFs, we propose that the membrane assembly or detachment of the VFs of cotonvirus particles involves or requires the function of the membranes of the Golgi apparatus, which is different from what has been observed for other icosahedral lineages. ...
Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and non-tailed mimiviruses has not yet been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae , excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae . Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than to the others. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and period of infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae . Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Mimivirinae .
Importance
We have isolated a new virus of an independent lineage belonging to the family Mimiviridae , subfamily Megavirinae , from the fresh water of a canal in Japan, named cotonvirus. In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae —lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are similar to those of tupanviruses, rather than the A, B, or C lineages, and other genomic features and host specificity of cotonvirus are similar to those of the latter than the former. These results suggest that cotonvirus is a unique virus, with chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.
... In the center of the factory, a small number of vesicles and individual fragments of membrane were seen. However, in this study, we did not observe any membrane vesicles in the space between the periphery and the center of the factory, which could have provided evidence for the membrane vesicles being the origin of virus membrane, as suggested for mimivirus [27]. Further, membrane fragments in the factory were small and did not have the acute curvature which would be expected of ruptured vesicles, as seen in the assembly of other NCLDVs. ...
African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis.
... Beside high-resolution structural studies on icosahedral NCLDVs, their morphogenesis was also intensively studied through a combination of various imaging techniques within the host cells (37,42,50,51,73). The viral assembly process of NCLDVs happens in the viral factories where mass reorganization of the host cell cytoskeleton and membranes occurs to coordinate viral genome replication and virion assembly. ...
... This capsid remains incomplete until the genome is packaged through a large portal (37). Similar viral assembly processes were also seen in Mimivirus (42) and ASFV (50), suggesting a general mechanism of their viral membrane formation and capsid assembly. ...
Nucleocytoplasmic large DNA viruses (NCLDVs) are a group of large viruses that infect a wide range of hosts, from animals to protists. These viruses are grouped together in NCLDV based on genomic sequence analyses. They share a set of essential genes for virion morphogenesis and replication. Most NCLDVs generally have large physical sizes while their morphologies vary in different families, such as icosahedral, brick, or oval shape, raising the question of the possible regulatory factor on their morphogenesis. The capsids of icosahedral NCLDVs are assembled from small building blocks, named capsomers, which are the trimeric form of the major capsid proteins. Note that the capsids of immature poxvirus are spherical even though they are assembled from capsomers that share high structural conservation with those icosahedral NCLDVs. The recently published high resolution structure of NCLDVs, Paramecium bursaria Chlorella virus 1 and African swine fever virus, described the intensive network of minor capsid proteins that are located underneath the capsomers. Among these minor proteins is the elongated tape measure protein (TmP) that spans from one icosahedral fivefold vertex to another. In this study, we focused on the critical roles that TmP plays in the assembly of icosahedral NCLDV capsids, answering a question raised in a previously proposed spiral mechanism. Interestingly, basic local alignment search on the TmPs showed no significant hits in poxviruses, which might be the factor that differentiates poxviruses and icosahedral NCLDVs in their morphogenesis.
... In addition to +RNA viruses, other viruses containing double-stranded RNA (Reoviruses) as well as DNA viruses (Poxvirus, Vaccina virus, African swine fever virus, Frog Virus 3 and Paramecium Bursaria Chlorella Virus, giant Mimivirus Acanthamoeba polyphaga) also induce massive host membrane rearrangements. Although less studied, those viruses also rely on the production of proteins modifying the curvature of the host membrane (Weisberg et al. 2017;Moss 2018, Mutsafi et al. 2013). The membrane-enveloped double stranded RNA bacteriophages from the cystoviridiae family, such as phage φ6, are the only known enveloped phages and are evolutionarily related to the +RNA eukaryotic virus picornavirus (Koonin, Dolja and Krupovic 2015). ...
Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering membrane proliferation share two major common features: 1) they promote the formation of highly curved membrane domains and 2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, inspiring new biotechnological applications.
... Giant viruses have notable differences in their replication cycles and structures of their virions. After the discovery of this group of viruses, studies have been conducted to increase the knowledge of their biology and host interaction through investigations related to the life cycle of these organisms [11,16,17]. This study presents an indepth description of the stages of the Orpheovirus life cycle, providing information about the entry, morphogenesis, release, structural characteristics and CPE triggered by this virus in its host cells. ...
Background:
After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated.
Methods:
In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays.
Results:
We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant.
Conclusions:
The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.
