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Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ion beam (FIB) milling-produced lamellae of mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., “molecular sociology”) of host and virus in 3D at two time points post-infection, enabling a detailed description of these condensates and a mechanistic understanding of MRV replication within them. Expanding over time, the condensate fashions host ribosomes at its periphery, and host microtubules, lipid membranes, and viral molecules in its interior, forming a 3D architecture that supports the dynamic processes of viral genome replication and capsid assembly. A total of six MRV assembly intermediates are identified inside the condensate: star core, empty and genome-containing cores, empty and full virions, and outer shell particle. Except for star core, these intermediates are visualized at atomic resolution by cryogenic electron microscopy (cryoEM) of cellular extracts. The temporal sequence and spatial rearrangement among these viral intermediates choreograph the viral life cycle within the condensates. Together, the molecular sociology of MRV-induced cellular condensate highlights the functional advantage of transient enrichment of molecules at the right location and time for viral replication. Virus-induced cellular condensates are poorly understood. Using cryoET and cryoEM, the authors visualized the 3D “molecular sociology” of host-virus interactions and choreographed mammalian reovirus assembly and replication within these condensates.

This study visualizes the interior of a dsRNA virus using cryo-electron microscopy, revealing the organization of the genome of cytoplasmic polyhedrosis virus together with its transcriptional enzyme complex in both quiescent and transcribing states. Genome packing in a dsRNA virus Genome packaging in double-stranded RNA viruses is poorly understood. Using direct electron-counting cryoelectron microscopy and asymmetric reconstruction, Hong Zhou and colleagues visualize in situ structures of the genome of insect cytoplasmic polyhedrosis virus (CPV) in quiescent and transcribing states. The structures reveal that each CPV capsid contains ten segmented dsRNAs, organized with ten transcriptional enzyme complexes in a specific, non-symmetric manner, with each dsRNA segment attached directly to a transcriptional enzyme complex. Viruses in the Reoviridae , like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC) 1 . By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

Double-stranded RNA (dsRNA) viruses possess a segmented dsRNA genome and a number of RNA-dependent RNA polymerases (RdRps) enclosed in a capsid. Until now, the precise structures of genomes and RdRps within the capsids have been unknown. Here we report the structures of RdRps and associated RNAs within nontranscribing and transcribing cypoviruses (NCPV and TCPV, respectively), using a combination of cryo–electron microscopy (cryo-EM) and a symmetry-mismatch reconstruction method. The RdRps and associated RNAs appear to exhibit a pseudo-D3 symmetric organization in both NCPV and TCPV. However, the molecular interactions between RdRps and the genomic RNA were found to differ in these states. Our work provides insight into the mechanisms of the replication and transcription in dsRNA viruses and paves a way for structural determination of lower-symmetry complexes enclosed in higher-symmetry structures.

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8–3.5 Å) RdRp–RNA in situ structures—representing quiescent, initiation, early elongation, elongation and abortive states—under seven experimental conditions of CPV. We observed the ‘Y’-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

Cytoplasmic polyhedrosis virus Cytoplasmic polyhedrosis virus (CPV) is a member of a large family of double-stranded RNA viruses, but it is unique in having a single shell capsid yet being fully capable of cell entry and mRNA transcription. The structure of this virus has now been determined by single-particle cryo-electron microscopy (cryoEM) to a resolution of 3.88 Å. The technique allows the polypeptide backbone to be traced without the need to make a crystal. The high-resolution structure shows how conformational switching is exploited to make railroad-like 'sliding' tracks for RNA packing and transcription and reveals an mRNA releasing hole coupled with distinctive capping machinery. With this and several other recent publications, cryo-electron microscopy underlines its credentials as a system capable of atomic-resolution in structural studies. This paper presents a high resolution structure of the cytoplasmic polyhedroasis virus (CPV) obtained by single particle cryo electron microscopy, and shows that the polypeptide backbone can be traced without the need of making a crystal. Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription 1 , 2 , 3 , 4 . Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation 5 , 6 . Here we report the three-dimensional structure of CPV at 3.88 Å resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of α-helices, the strand separation in β-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-β-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.

Many viruses utilize trimeric spikes to gain entry into host cells. However, without in situ structures of these trimeric spikes, a full understanding of this dynamic and essential process of viral infections is not possible. Here we present four in situ and one isolated cryoEM structures of the trimeric spike of the cytoplasmic polyhedrosis virus, a member of the non-enveloped Reoviridae family and a virus historically used as a model in the discoveries of RNA transcription and capping. These structures adopt two drastically different conformations, closed spike and opened spike, which respectively represent the penetration-inactive and penetration-active states. Each spike monomer has four domains: N-terminal, body, claw, and C-terminal. From closed to opened state, the RGD motif-containing C-terminal domain is freed to bind integrins, and the claw domain rotates to expose and project its membrane insertion loops into the cellular membrane. Comparison between turret vertices before and after detachment of the trimeric spike shows that the trimeric spike anchors its N-terminal domain in the iris of the pentameric RNA-capping turret. Sensing of cytosolic S-adenosylmethionine (SAM) and adenosine triphosphate (ATP) by the turret triggers a cascade of events: opening of the iris, detachment of the spike, and initiation of endogenous transcription. Zhang and Cui et al. present in situ cryoEM structures of the trimeric spike of cytoplasmic polyhedrosis virus in both open and close conformations, and demonstrate that spike detachment from the capsid is triggered by the presence of SAM and ATP.

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7–98.3% nucleotide identity, a 96.4–100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

Summary Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini‐organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high‐pressure frozen and freeze‐substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule‐dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule‐independent strain, progeny virions lacked organisation. Conversely to the microtubule‐dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome‐less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule‐dependent strain resulted in a significant increase of genome‐less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.

Tough cubes Insect viruses that produce infectious polyhedra — crystals encapsulating thousands of virus particles — are widespread and important. The polyhedra microcrystals are remarkably stable, which can cause disease persistence, threatening silkworm cocoon harvests for instance. The molecular structure of one of these crystals has now been determined. This is a major technical feat in protein X-ray microcrystallography, as these viruses are the smallest protein crystals ever used to determine atomic structure. The study reveals robust polyhedra that could be useful as delivery capsules for biopesticides and for nanobiotechnology applications such as microarrays. Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra 1 , 2 . The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides 3 , 4 . Although polyhedra have been extensively characterized since the early 1900s 5 , their atomic organization remains elusive 6 . Here we describe the 2 Å crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5–12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination 7 . We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen–antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications 8 such as microarrays 9 and biopesticides 4 .