FIG 8 - uploaded by Benjamin G Kopek
Content may be subject to copyright.
Polymerase independence and dependence of BMV and FHV spherule membrane rearrangements and replication complex assembly. (A) BMV spherule generation by multifunctional BMV RNA replication factor 1a (blue). In the absence of BMV RNA-dependent RNA polymerase 2a Pol , 1a localizes to ER membranes, self-interacts, and induces the formation of 70-nm invaginations or spherules (39, 40, 51). In a separable subsequent reaction dependent on the activity of the C-proximal 1a NTPase/helicase domain, 1a transfers genomic RNA (red line) to the spherule interior (61). (B) In the presence of 1a and 2a Pol (yellow), 1a also recruits 2a Pol to ER membranes and directs spherule formation and genomic RNA recruitment as in panel A. 2a Pol then synthesizes negative-strand RNA (dashed black line) that is retained in the spherule and repeatedly used as a template to synthesize new positive strands. (C) Protein A (green), the sole FHV-encoded RNA replication factor, localizes to mitochondrial outer membranes (34) and self-interacts (16) but does not induce spherule formation unless replication-competent FHV RNA templates are present and protein A's RNA polymerase domain is active (Fig. 2 to 7). Active-site mutations abolishing protein A polymerase activity or deletion of 3 RNA replication signals still allow protein A to recognize specific 5-proximal elements in viral RNA templates and recruit them to mitochondrial membranes (59, 60) but block RNA synthesis and invagination of FHV spherules (Fig. 7). See Discussion for additional details, including the role of FHV RNA interference suppressor B2.
Source publication
Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ∼50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+...
Contexts in source publication
Context 1
... study reveals that while FHV and some other ()RNA viruses such as BMV replicate their RNA genomes in mem- brane compartments of similar architecture, they generate these compartments by very different RNA-and polymerase- dependent or -independent pathways (Fig. 8). Below, we dis- cuss the relationships of these findings to other features of nodavirus RNA replication, to other viruses, and to viral in- teraction with host innate immune ...
Context 2
... such similarities, we find that FHV and BMV mem- brane spherules are generated by highly distinct mechanisms. BMV 1a is the sole viral component required to induce BMV replication spherules, with no requirement for viral RNA tem- plates or the BMV RNA polymerase, 2a pol (51) (Fig. 8A and B). Similarly, for many other ()RNA viruses, one or two nonpolymerase RNA replication proteins suffice to induce the membrane rearrangements associated with RNA replication ...
Context 3
... in spherule architecture suggested that FHV protein A was likely sufficient to form spherules. However, in this study, we found that protein A, although the only viral protein required, was insufficient for spherule formation (Fig. 2 and 4). Forming FHV spherules also required functional viral RNA templates and protein A's RNA polymerase activity (Fig. 8C). Multiple results indicated that RNA synthesis was required. In the pres- ence of WT protein A, spherule formation was supported by genomic RNA1 or subgenomic RNA3, which are both repli- cation templates (Fig. 3 and 6), but was blocked by the deletion of 3 replication signals (Fig. 7). Similarly, in the presence of WT FHV RNA ...
Context 4
... to translocate RNA into a preformed spherule, FHV may be obliged to trigger the formation of its replication compart- ments around its RNA templates or replication intermediates. Coupling such membrane rearrangements with RNA synthesis further ensures that a replication-competent viral template is enclosed within the replication compartment (Fig. ...
Context 5
... innate immune responses such as RNAi (1). However, our results show that formation of FHV spherules requires FHV RNA synthesis. Thus, there must be a stage in the FHV rep- lication cycle, prior to spherule formation, when dsRNA rep- lication intermediates are potentially accessible to the siRNA- producing Dicer nuclease and other RNAi components (Fig. 8). These results provide an explanation for recent findings that virus-specific siRNAs produced in FHV infection are de- rived from both strands, implying that dsRNA replication in- termediates are the predominant source of these siRNAs (5,20,58). Once spherules are formed, they may contribute to protecting viral replication ...
Similar publications
Unlabelled:
Viruses with positive-strand RNA genomes amplify their genomes in replication complexes associated with cellular membranes. Little is known about the mechanism of replication complex formation in cells infected with Nodamura virus. This virus is unique in its ability to lethally infect both mammals and insects. In mice and in larvae of...
Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs
(siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based
antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in...
Citations
... The biogenesis of DMV occurs during infection with numerous +RNA viruses. Recently, the role of viral nsps in DMV biosynthesis has been considerably advanced, yet the key mechanisms or host factors of DMV production remain under investigation (132)(133)(134). Out of the nidoviruses, coronaviruses and arteritiviruses have been extensively researched, while mesoniviruses and roniviruses have received comparatively less attention (5,135). ...
During infection, positive-stranded RNA causes a rearrangement of the host cell membrane, resulting in specialized membrane structure formation aiding viral genome replication. Double-membrane vesicles (DMVs), typical structures produced by virus-induced membrane rearrangements, are platforms for viral replication. Nidoviruses, one of the most complex positive-strand RNA viruses, have the ability to infect not only mammals and a few birds but also invertebrates. Nidoviruses possess a distinctive replication mechanism, wherein their nonstructural proteins (nsps) play a crucial role in DMV biogenesis. With the participation of host factors related to autophagy and lipid synthesis pathways, several viral nsps hijack the membrane rearrangement process of host endoplasmic reticulum (ER), Golgi apparatus, and other organelles to induce DMV formation. An understanding of the mechanisms of DMV formation and its structure and function in the infectious cycle of nidovirus may be essential for the development of new and effective antiviral strategies in the future.
... Interestingly, several reports have shown that doublemembrane vesicles (DMVs) are associated with positive-sense ssRNA (+ RNA) virus infections in eukaryotic cells due to the hijacking of secretory pathways and the induction of membrane remodelling [102]. The ectopic expression of certain non-structural viral proteins, such as transmembrane or membrane-tethering domains, or viral RNAs has been suggested to play crucial roles in DMV biogenesis [103,104]. ...
Extracellular vesicles (EVs) have garnered significant interest in the field of biomedical science due to their potential applications in therapy and diagnosis. These vesicles participate in cell-to-cell communication and carry a diverse range of bioactive cargo molecules, such as nucleic acids, proteins, and lipids. These cargoes play essential roles in various signaling pathways, including paracrine and endocrine signaling. However, our understanding of the morphological and structural features of EVs is still limited. EVs could be unilamellar or multilamellar or even multicompartmental structures. The relative proportions of these EV subtypes in biological fluids have been associated with various human diseases; however, the mechanism remains unclear. Cryo-electron microscopy (cryo-EM) holds great promise in the field of EV characterization due to high resolution properties. Cryo-EM circumvents artifacts caused by fixation or dehydration, allows for the preservation of native conformation, and eliminates the necessity for staining procedures. In this review, we summarize the role of EVs biogenesis and pathways that might have role on their structure, and the role of cryo-EM in characterization of EVs morphology in different biological samples and integrate new knowledge of the alterations of membranous structures of EVs which could be used as biomarkers to human diseases.
... In the absence of other viral components, protein A targets itself to OMMs (17,18). Protein A specifically recognizes viral RNA templates and recruits them to the OMM (18,19), then synthesizes (−)RNA, and captures the resulting dsRNA product in an RC vesicle newly invaginated on the OMM (20). When trapped on OMMs in its pre-RNA synthesis state by polymerase mutation or absence of a replicable FHV RNA template, protein A fails to induce RNA replication vesicles and causes extensive, close zippering of adjacent mitochondria (20). ...
... Protein A specifically recognizes viral RNA templates and recruits them to the OMM (18,19), then synthesizes (−)RNA, and captures the resulting dsRNA product in an RC vesicle newly invaginated on the OMM (20). When trapped on OMMs in its pre-RNA synthesis state by polymerase mutation or absence of a replicable FHV RNA template, protein A fails to induce RNA replication vesicles and causes extensive, close zippering of adjacent mitochondria (20). The spaces between such zippered mitochondria contain regular OMM-linked structures with ~20 nm periodicity (20). ...
... When trapped on OMMs in its pre-RNA synthesis state by polymerase mutation or absence of a replicable FHV RNA template, protein A fails to induce RNA replication vesicles and causes extensive, close zippering of adjacent mitochondria (20). The spaces between such zippered mitochondria contain regular OMM-linked structures with ~20 nm periodicity (20). Thus, prior to RNA synthesis, protein A induces OMM-linked complexes with dimensions similar to the mature crowns of active RCs, but with dramatically different interaction properties. ...
Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesi-cles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold sym-metric “crown” at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a “proto-crown” precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding “legs.” The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.
... As noted in the Introduction, when nodavirus RNA replication is blocked by omitting the RNA template or mutating the template or Pol active site, protein A assembles on OMMs in ordered arrangements with interactive properties distinct from the crowns of full FHV infection (20). To determine the structure of these "pre-replication" protein A assemblies, we used recombinant baculoviruses AcR1 and AcR1Δ3', kindly provided by Dr. Anette Schneemann (21). ...
... Forming nodavirus RNA replication vesicles requires protein A, a functional RNA template and RNA synthesis (20), suggesting that the vesicle is generated by being filled with the dsRNA product of (-)RNA synthesis (9,12). The resulting replication vesicle, pressurized by negatively charged dsRNA, is held together at the neck by the mature crown, which depends on its dual rings of membrane interaction sites in the upper crown legs as well as the lower proto-crown floor to closely constrain and stabilize the curved membrane neck ( Fig. 3A and (10)). ...
... Beyond (+)RNA synthesis and in addition to their structural roles, the Pol domains in the proto-crown or mature crown central turrets likely function in other required Pol functions, including RNA template recruitment and (-)RNA synthesis (2,19,20), and possibly bridging interactions to downstream processes like translation and encapsidation (see next section). ...
Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric “crown” at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryo-electron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multi-domain viral RNA replication protein A. Each ring contains an ~ 19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a “proto-crown” precursor that assembles prior to RNA template recruitment, RNA synthesis and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding “legs.” The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and non-symmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility and evolutionary plasticity and new insights into (+)RNA virus replication and control.
Significance
Positive-strand RNA viruses - including coronaviruses, alphaviruses, flaviviruses and many other medically and economically important pathogens - replicate their RNA genomes by virus-encoded machinery that has been poorly characterized. Using an advanced nodavirus model, we identify a major precursor in RNA replication complex assembly and show it to be a 12-mer ring of viral RNA replication protein A, whose single particle cryo-EM structure reveals functional features of its membrane interaction, assembly, polymerase and RNA capping domains. We further show that fully functional RNA replication complexes acquire a second 12-mer ring of protein A in alternate conformation atop the first, and a central density likely to represent another polymerase conformation. These findings provide strong foundations for understanding, controlling and beneficially using such viruses.
... As noted in the Introduction, FHV protein A localizes to OMMs where it recruits viral RNA templates, induces spherule RCs and provides essential enzymatic functions for RNA synthesis [14,16,17,20]. In keeping with these functions, immunogold labeling shows that protein A is present in the protein crowns of FHV RC spherules [23] as a repeated, 12-fold symmetric component [24]. ...
... Of the remaining nodaviral proteins, B1 is not only dispensable for RNA replication but localizes to the nucleus and not at the cytoplasmic sites of FHV RNA replication [10,11]. Moreover, RNA1 alone can induce RNA replication, showing that RNA2 and its encoded capsid protein are not required for RNA1 replication and subgenomic RNA3 transcription [20]. RNAi inhibitor protein B2 likewise is dispensable if RNAi is suppressed by other means [20]. ...
... Moreover, RNA1 alone can induce RNA replication, showing that RNA2 and its encoded capsid protein are not required for RNA1 replication and subgenomic RNA3 transcription [20]. RNAi inhibitor protein B2 likewise is dispensable if RNAi is suppressed by other means [20]. However, RC crown structures have not been studied under these conditions to date and the B2 and capsid proteins remain prime candidates for participating in wildtype crowns, since nodavirus virion assembly has been linked to RNA replication and virions localize closely with viral RNA replication compartments [30,31], and B2 has been reported to interact with protein A in infected cells [32]. ...
Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are “crowned” by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.
... Moreover, the term 'antiterminator', describing the specific activity of this protein in genome replication and replication organelle biogenesis, is not typically used in positivesense RNA viruses and, instead, these proteins are referred to as 'replication organelle biogenesis' proteins [16,17]. However, the vesicular nature of their replication organelles does not preclude their formation via phase separation [59][60][61][62][63]. Rather, the replication organelle biogenesis protein, hereon referred to as an antiterminator, typically either contains an amphipathic or transmembrane domain that directly targets it to a cellular membrane (e.g., the endoplasmic reticulum or Golgi complex), or it interacts directly with another viral replication protein that is membrane associated [16,17,59,60,[64][65][66][67]. In both positive-and negative-sense RNA viruses, antiterminator interactions are typically nucleated by high-affinity binding sites on the viral RNA, and condensation of the viral RNA(s) is likely promoted by low-affinity interactions across the remainder of the viral genome ( Figure 2B) [60,64,[67][68][69][70][71]. ...
... Antiterminator-mediated replication organelle biogenesis generally requires RNA and, at least for some positive-sense RNA viruses, the size of the replication organelle is defined by the length of the viral genomic RNA [28,29,66,67,70,[80][81][82][83][84]. Notably, in some cases, cellular expression of specific viral nonstructural proteins in the absence of infection was shown to be sufficient for replication organelle biogenesis; thus, it was assumed that RNA was not required for this process [84][85][86][87][88]. ...
... However, in the absence of viral substrate RNA(s), it is possible that viral antiterminator protein(s) can condense cellular or plasmid-derived RNAs to form replication organelle-like structures, as previously shown for the antiterminator protein (1a) of brome mosaic virus [64,87]. This would explain the heterogenous size and nature of the replication organelle-like structures observed when viral nonstructural proteins are expressed in isolation, in contrast to the relatively uniform size distribution observed during infection (at least in positive-sense RNA viruses), and is consistent with the idea that RNA is important for viral replication organelle biogenesis [66,67,[81][82][83][84]. By contrast, the rotavirus (a dsRNA virus) antiterminator (NSP2) and phosphoprotein (NSP5) have been demonstrated to phase separate in the absence of RNA in vitro; however, this process is likely facilitated by RNA within cells [6]. ...
Viruses compartmentalize their replication and assembly machinery to both evade detection and concentrate the viral proteins and nucleic acids necessary for genome replication and virion production. Accumulating evidence suggests that diverse RNA and DNA viruses form replication organelles and nucleocapsid assembly sites using phase separation. In general, the biogenesis of these compartments is regulated by two types of viral protein, collectively known as antiterminators and nucleocapsid proteins, respectively. Herein, we discuss how RNA viruses establish replication organelles and nucleocapsid assembly sites, and the evidence that these compartments form through phase separation. While this review focuses on RNA viruses, accumulating evidence suggests that all viruses rely on phase separation and form biomolecular condensates important for completing the infectious cycle.
... These mitochondrial membrane vesicles have an average diameter of ~50 nm and, like other In-vROs, are connected to the cytosol through a small pore-like opening. In addition to Protein A, the formation of FHV vROs requires both the presence of a viral genome and an active RNA-dependant RNA polymerase (RdRp), indicating that initial rounds of genome replication or negative strand synthesis play a role in biogenesis [27]. ...
Virus infection is a process that requires combined contributions from both virus and host factors. For this process to be efficient within the crowded host environment, viruses have evolved ways to manipulate and reorganize host structures to produce cellular microenvironments. Positive-strand RNA virus replication and assembly occurs in association with cytoplasmic membranes, causing a reorganization of these membranes to create microenvironments that support viral processes. Similarities between virus-induced membrane domains and cellular organelles have led to the description of these structures as virus replication organelles (vRO). Electron microscopy analysis of vROs in positive-strand RNA virus infected cells has revealed surprising morphological similarities between genetically diverse virus species. For all positive-strand RNA viruses, vROs can be categorized into two groups: those that make invaginations into the cellular membranes (In-vRO), and those that cause the production of protrusions from cellular membranes (Pr-vRO), most often in the form of double membrane vesicles (DMVs). In this review, we will discuss the current knowledge on the structure and biogenesis of these two different vRO classes as well as comparing morphology and function of vROs between various positive-strand RNA viruses. Finally, we will discuss recent studies describing pharmaceutical intervention in vRO formation as an avenue to control virus infection.
... This is also the case for the nsp6 protein of SARS-CoV [20], the NS1-2 and NS3 proteins of noroviruses [9] and the nsp5 protein of arteriviruses [26]. Moreover, other models of + RNA viruses have shown that replicating viral RNA is also a major player in the biogenesis of virus-induced membrane rearrangements [27]. ...
Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.
... To date, alphanodaviruses have not been reported to cause significant threats to the global economy [28]. Due to their small genome size and efficient replication in many hosts, several alphanodaviruses, especially Flock House virus (FHV), have been modified and successfully used as excellent models for the studies of positive-strand RNA virus infections in their hosts [29][30][31][32]. ...
... Due to their relatively small genomes, alphanodaviruses, especially FHV and NoV, have been modified and used as models in many studies [28][29][30][31][32]. They have also been used as viral vectors in basic and applied research, including heterologous protein expression and gene silencing [1,[68][69][70][71]. ...
Nodaviruses are small bipartite RNA viruses and are considered animal viruses. Here, we identified two novel noda-like viruses (referred to as rice-associated noda-like virus 1 (RNLV1) and rice-associated noda-like virus 2 (RNLV2)) in field-collected rice plants showing a dwarfing phenotype through RNA-seq. RNLV1 genome consists of 3335 nt RNA1 and 1769 nt RNA2, and RNLV2 genome consists of 3279 nt RNA1 and 1525 nt RNA2. Three conserved ORFs were identified in each genome of the two novel viruses, encoding an RNA-dependent RNA polymerase, an RNA silencing suppressor, and a capsid protein, respectively. The results of sequence alignment, protein domain prediction, and evolutionary analysis indicate that these two novel viruses are clearly different from the known nodaviruses, especially the CPs. We have also determined that the B2 protein encoded by the two new noda-like viruses can suppress RNA silencing in plants. Two reverse genetic systems were constructed and used to show that RNLV1 RNA1 can replicate in plant cells and RNLV1 can replicate in insect Sf9 cells. We have also found two unusual peptidase family A21 domains in the RNLV1 CP, and RNLV1 CP can self-cleave in acidic environments. These findings provide new knowledge of novel nodaviruses.
... Protein A also recruits FHV genomic RNA templates to the outer mitochondrial membrane by interaction of its RdRp domain with cis signals that in FHV genomic RNA1 reside in nucleotides 68-205 (22, 23). In a process closely linked to RNA synthesis from such templates, protein A then induces invagination of the characteristic ∼60to 70-nm spherule vesicles of its RCs (24). ...
... Given these new revelations on FHV RC and crown structure, the tight linkage of FHV spherule formation to viral RNA synthesis (24) suggests that the spherule RC may be formed during (−)RNA synthesis by inflating the vesicle with the dsRNA product, like blowing up a balloon (12). A similar model was proposed among other alternatives for Semliki Forest alphavirus (27). ...
Positive-strand RNA viruses, the largest genetic class of eukaryotic viruses, include coronaviruses and many other established and emerging pathogens. A major target for understanding and controlling these viruses is their genome replication, which occurs in virus-induced membrane vesicles that organize replication steps and protect double-stranded RNA intermediates from innate immune recognition. The structure of these complexes has been greatly illuminated by recent cryo-electron microscope tomography studies with several viruses. One key finding in diverse systems is the organization of crucial viral RNA replication factors in multimeric rings or crowns that among other functions serve as exit channels gating release of progeny genomes to the cytosol for translation and encapsidation. Emerging results suggest that these crowns serve additional important purposes in replication complex assembly, function, and interaction with downstream processes such as encapsidation. The findings provide insights into viral function and evolution and new bases for understanding, controlling, and engineering positive-strand RNA viruses.
Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
