Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 Å by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase λ3, were then fitted into the density map. Each copy of λ3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein λ1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of λ3 is bound per vertex. λ3 is furthermore oriented with its transcript exit channel facing a small channel through the λ1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein λ2.
Among members of the genus Orthoreovirus, family Reoviridae, a group of non-enveloped viruses with genomes comprising ten segments of double-stranded RNA, only the ‘non-fusogenic’ mammalian orthoreoviruses (MRVs) have been studied to date by electron cryomicroscopy and three-dimensional image reconstruction. In addition to MRVs, this genus comprises other species that induce syncytium formation in cultured cells, a property shared with members of the related genus Aquareovirus. To augment studies of these ‘fusogenic’ orthoreoviruses, we used electron cryomicroscopy and image reconstruction to analyze the virions of a fusogenic avian orthoreovirus (ARV). The structure of the ARV virion, determined from data at an effective resolution of 14.6 Å , showed strong similarities to that of MRVs. Of particular note, the ARV virion has its pentameric λ-class core turret protein in a closed conformation as in MRVs, not in a more open conformation as reported for aquareovirus. Similarly, the ARV virion contains 150 copies of its monomeric σ-class core-nodule protein as in MRVs, not 120 copies as reported for aquareovirus. On the other hand, unlike that of MRVs, the ARV virion lacks ‘hub-and-spokes’ complexes within the solvent channels at sites of local sixfold symmetry in the incomplete T = 13l outer capsid. In MRVs, these complexes are formed by C-terminal sequences in the trimeric µ-class outer-capsid protein, sequences that are genetically missing from the homologous protein of ARVs. The channel structures and C-terminal sequences of the homologous outer-capsid protein are also genetically missing from aquareoviruses. Overall, the results place ARVs between MRVs and aquareoviruses with respect to the highlighted features.
Orthoreovirus; Reovirus; Reoviridae; Virus structure; Electron cryomicroscopy
Cell entry by nonenveloped animal viruses requires membrane penetration without membrane fusion. The reovirus penetration agent is the outer-capsid protein, μ1. The structure of μ1, complexed with its “protector” protein, σ3, and the fit of this μ13σ33 heterohexameric complex into the cryoEM image of an intact virion, reveal molecular events essential for viral penetration. Autolytic cleavage divides μ1 into myristoylated μ1N and μ1C. A long hydrophobic pocket can receive the myristoyl group. Dissociation of μ1N, linked to a major conformational change of the entire μ1 trimer, must precede myristoyl-group insertion into the cellular membrane. A myristoyl switch, coupling exposure of the fatty acid chain, autolytic cleavage of μ1N, and long-range molecular rearrangement of μ1C, thus appears to be part of the penetration mechanism.
Reovirus is a useful model for addressing the molecular basis of membrane penetration by one of the larger nonenveloped animal viruses. We now report the structure of the reovirus virion at 7.0 Å resolution as obtained by electron cryomicroscopy and three-dimensional image reconstruction. Several features of the myristoylated outer capsid protein μ1, not seen in a previous X-ray crystal structure of the μ1-σ3 heterohexamer, are evident in the virion. These features appear to be important for stabilizing the outer capsid, regulating the conformational changes in μ1 that accompany perforation of target membranes, and contributing directly to membrane penetration during cell entry.
The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves −2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date.
Trichomonas vaginalis causes ~250 million new cases of sexually transmitted disease each year worldwide and is associated with serious complications, including premature birth and increased transmission of other pathogens, including HIV. It is an extracellular parasite that, in turn, commonly hosts infections with double-stranded RNA (dsRNA) viruses, trichomonasviruses, which appear to exacerbate disease through signaling of immunoinflammatory responses by human epithelial cells. Here we report the first three-dimensional structure of a trichomonasvirus, which is also the first such structure of any protozoan dsRNA virus; show that it has unusually wide channels at the capsid vertices, with potential for releasing the viral genome and promoting dsRNA-dependent responses by human cells; and provide evidence that it uses −2 ribosomal frameshifting, an uncommon mechanism, to translate its RNA polymerase in fusion with its capsid protein. These findings provide both mechanistic and translational insights concerning the role of trichomonasviruses in aggravating disease attributable to T. vaginalis.
Trichomonas vaginalis, which causes the most common nonviral sexually transmitted disease worldwide, is itself commonly infected by nonsegmented double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae. To date, cDNA sequences of one or more strains of each of three trichomonasvirus species have been reported, and gel electrophoresis showing several different dsRNA molecules obtained from a few T. vaginalis isolates has suggested that more than one virus strain might concurrently infect the same parasite cell. Here, we report the complete cDNA sequences of 3 trichomonasvirus strains, one from each of the 3 known species, infecting a single, agar-cloned clinical isolate of T. vaginalis, confirming the natural capacity for concurrent (in this case, triple) infections in this system. We furthermore report the complete cDNA sequences of 11 additional trichomonasvirus strains, from 4 other clinical isolates of T. vaginalis. These additional strains represent the three known trichomonasvirus species, as well as a newly identified fourth species. Moreover, 2 of these other T. vaginalis isolates are concurrently infected by strains of all 4 trichomonasvirus species (i.e., quadruple infections). In sum, the full-length cDNA sequences of these 14 new trichomonasviruses greatly expand the existing data set for members of this genus and substantiate our understanding of their genome organizations, protein-coding and replication signals, diversity, and phylogenetics. The complexity of this virus-host system is greater than has been previously well recognized and suggests a number of important questions relating to the pathogenesis and disease outcomes of T. vaginalis infections of the human genital mucosa.
Virally induced structures called viral factories form throughout the cytoplasm of cells infected with mammalian orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein μNS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. μNS also associates with MRV core particles; the core proteins μ2, λ1, λ2, λ3, and σ2; and the RNA-binding nonstructural protein σNS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of μNS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of μNS are necessary for associations with core particles and necessary and sufficient for associations with the proteins μ2, λ1, λ2, σ2, and σNS. We also found that only the λ3 protein associates with the carboxyl-terminal one-third of μNS and that viral RNA is synthesized within viral factories. These results suggest that μNS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV infection.
Family Reoviridae, subfamily Spinareovirinae, includes nine current genera. Two of these genera, Aquareovirus and Orthoreovirus, comprise members that are closely related and consistently share nine homologous proteins. Orthoreoviruses have 10 dsRNA genome segments and infect reptiles, birds, and mammals, whereas aquareoviruses have 11 dsRNA genome segments and infect fish. Recently, the first 10-segmented fish reovirus, piscine reovirus (PRV), has been identified and shown to be phylogenetically divergent from the 11-segmented viruses constituting genus Aquareovirus. We have recently extended results for PRV by showing that it does not encode a fusion-associated small transmembrane (FAST) protein, but does encode an outer-fiber protein containing a long N-terminal region of predicted α-helical coiled coil. Three recently characterized 11-segmented fish reoviruses, obtained from grass carp in China and sequenced in full, are also divergent from the viruses now constituting genus Aquareovirus, though not to the same extent as PRV. In the current study, we reexamined the sequences of these three recent isolates of grass carp reovirus (GCRV)–HZ08, GD108, and 104–for further clues to their evolution relative to other aqua- and orthoreoviruses. Structure-based fiber motifs in their encoded outer-fiber proteins were characterized, and other bioinformatics analyses provided evidence against the presence of a FAST protein among their encoded nonstructural proteins. Phylogenetic comparisons showed the combination of more distally branching, approved Aquareovirus and Orthoreovirus members, plus more basally branching isolates GCRV104, GCRV-HZ08/GD108, and PRV, constituting a larger, monophyletic taxon not suitably recognized by the current taxonomic hierarchy. Phylogenetics also suggested that the last common ancestor of all these viruses was a fiber-encoding, nonfusogenic virus and that the FAST protein family arose from at least two separate gain-of-function events. In addition, an apparent evolutionary correlation was found between the gain or loss of NS-FAST and outer-fiber proteins among more distally branching members of this taxon.
Four-dimensional live-cell imaging is combined with single-particle tracking to identify key steps in polarized epithelium cell entry by the prototype enteric virus reovirus.
Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin–Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.
Filamentous fungus Fusarium poae is a worldwide cause of the economically important disease Fusarium head blight of cereal grains. The fungus is itself commonly infected with a bisegmented dsRNA virus from the family Partitiviridae. For this study, we determined the structure of partitivirus Fusarium poae virus 1 (FpV1) to a resolution of 5.6 Å or better by electron cryomicroscopy and three-dimensional image reconstruction. The main structural features of FpV1 are consistent with those of two other fungal partitiviruses for which high-resolution structures have been recently reported. These shared features include a 120-subunit T=1 capsid comprising 60 quasisymmetrical capsid protein dimers with both shell and protruding domains. Distinguishing features are evident throughout the FpV1 capsid, however, consistent with its more massive subunits and its greater phylogenetic divergence relative to the other two structurally characterized partitiviruses. These results broaden our understanding of conserved and variable elements of partitivirus structure, as well as that of picobirnavirus, and support the suggestion that a phylogenetic subcluster of partitiviruses closely related to FpV1 should constitute a separate taxonomic genus.
Capsid protein; Cryoelectron microscopy; dsRNA virus; Electron cryomicroscopy; Fungal virus; Virus structure
The family Totiviridae includes a number of viruses with monosegmented dsRNA genomes and isometric virions that infect either fungi or a number of medically important protozoan parasites such as Leishmania and Giardia. A new genus, Trichomonasvirus, was recently proposed for this family. Its name is based on the genus of its host organism, Trichomonas vaginalis, a protozoan parasite that colonizes the human genitourinary mucosa and is the most common non-viral sexually transmitted infection in the world. The type species of this new genus is Trichomonas vaginalis virus 1. Distinguishing characteristics of the new genus include infection of a human sexually transmitted parasite, stable mixed infection with more than one distinct Trichomonasvirus species, and sequence-based phylogenetic divergence that distinguishes it from all other family members.
Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300–450 Å in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination–reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 Å, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a “T = 2” icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses including, Leishmania RNA virus 1, to be similar as well.
Of the known dsRNA fungal viruses, the best characterized is Saccharomyces cerevisiae virus L-A (ScV-L-A), prototype of the genus Totivirus, family Totiviridae. Until the current study, there have been no subnanometer structures of dsRNA fungal viruses from the genus Victorivirus, which is the largest in family Totiviridae. The 3D cryo-reconstruction presented here of prototype victorivirus Helminthosporium victoriae virus 190S (HvV190S) approaches 7-Å resolution and shows the asymmetric unit of the capsid is a dimer comprising two, chemically identical coat-protein subunits organized in a so called “T = 2” lattice. These HvV190S subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses, including Leishmania RNA virus 1, to be similar as well.
The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV nonstructural protein, μNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that μNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by μNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of μNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, μNS. These results demonstrate μNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based in μNS-mediated sequestration.
adaptor; clathrin; membrane trafficking; reovirus; viral factories
The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus μ1 protein, encoded by the M2 gene, was sufficient to induce apoptosis in transfected cells. We also found that μ1 localized to lipid droplets, endoplasmic reticulum, and mitochondria in both transfected cells and infected cells. Two small regions encompassing amphipathic α-helices within a carboxyl-terminal portion of μ1 were necessary for efficient induction of apoptosis and association with lipid droplets, endoplasmic reticulum, and mitochondria in transfected cells. Induction of apoptosis by μ1 and its association with lipid droplets and intracellular membranes in transfected cells were abrogated when μ1 was coexpressed with σ3, with which it is known to coassemble. We propose that μ1 plays a direct role in the induction of apoptosis in infected cells and that this property may relate to the capacity of μ1 to associate with intracellular membranes. Moreover, during reovirus infection, association with σ3 may regulate apoptosis induction by μ1.
Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.
Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein μNS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with μNS. We found that all three of these proteins—λ1, λ2, and σ2—localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with μNS, on the other hand, each core surface protein colocalized with μNS in globular inclusions, supporting the initial hypothesis. We also found that λ1, λ2, and σ2 each localized to filamentous inclusions formed upon the coexpression of μNS and μ2, a structurally minor core protein that associates with microtubules. The first 40 residues of μNS, which are required for association with μ2 and the RNA-binding nonstructural protein σNS, were not required for association with any of the three core surface proteins. When coexpressed with μ2 in the absence of μNS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with μ2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of μNS that had been preformed in those cells, providing evidence that μNS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins μNS and μ2 in this process.
Baboon reovirus (BRV) is a member of the fusogenic subgroup of orthoreoviruses. Unlike most other members of its genus, BRV lacks S-segment coding sequences for the outer fiber protein that binds to cell surface receptors. It shares this lack with aquareoviruses, which constitute a related genus and are also fusogenic. We used electron cryomicroscopy and three-dimensional image reconstruction to determine the BRV virion structure at 9.0-Å resolution. The results show that BRV lacks a protruding fiber at its icosahedral 5-fold axes or elsewhere. The results also show that BRV is like nonfusogenic mammalian and fusogenic avian orthoreoviruses in having 150 copies of the core clamp protein, not 120 as in aquareoviruses. On the other hand, there are no hub-and-spoke complexes attributable to the outer shell protein in the P2 and P3 solvent channels of BRV, which makes BRV like fusogenic avian orthoreoviruses and aquareoviruses but unlike nonfusogenic mammalian orthoreoviruses. The outermost “flap” domains of the BRV core turret protein appear capable of conformational variability within the virion, a trait previously unseen among other ortho- and aquareoviruses. New cDNA sequence determinations for the BRV L1 and M2 genome segments, encoding the core turret and outer shell proteins, were helpful for interpreting the structural features of those proteins. Based on these findings, we conclude that the evolution of ortho- and aquareoviruses has included a series of discrete gains or losses of particular components, several of which cross taxonomic boundaries. Gain or loss of adhesion fibers is one of several common themes in double-stranded RNA virus evolution.
The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.
Here we describe the protein interaction platform assay, a method for identifying interacting proteins in Saccharomyces cerevisiae. This assay relies on the reovirus scaffolding protein μNS, which forms large focal inclusions in living cells. When a query protein is fused to μNS and potential interaction partners are fused to a fluorescent reporter, interactors can be identified by screening for yeast that display fluorescent foci.
The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the μ1 protein, each complexed with three copies of the protector protein σ3. Conformational changes in μ1 following the proteolytic removal of σ3 lead to release of the myristoylated N-terminal cleavage fragment μ1N and ultimately to membrane penetration. The μ1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant μ1 trimers and synthetic μ1N peptides with both RBCs and liposomes. The μ1 trimer mediates hemolysis and liposome disruption under conditions that promote the μ1 conformational change, and mutations that inhibit μ1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free μ1 trimer. Autolytic cleavage to form μ1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that μ1N cleavage is not required when steric barriers are removed. Synthetic myristoylated μ1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of β-strand secondary structure, suggest a model in which μ1N β-hairpins assemble in the membrane to form a β-barrel pore.
The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps.
Rotavirus RNA-dependent RNA polymerase, VP1, catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3′ end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 Å resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus λ3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3′ sequence. Well-defined interactions with these bases position the RNA so that its 3′ end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3′ end recognition selects rotavirus RNA for packaging and that VP2 activates the auto-inhibited VP1/RNA complex to coordinate packaging and genome replication.
Genome replication of mammalian orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein μNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, μNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for μNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (λ1, λ2, λ3, μ2, and σ2) and nonstructural protein σNS, can associate with μNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in μNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log10 values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement μNS expression and to rescue infectious yields by ~100-fold. We next used μNS mutants specifically defective at forming factory-matrix structures to show that this function of μNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of μNS prevented rescue of viral growth while causing μNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of μNS, designed to represent μNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of μNS known to be involved in both μ2 and σNS association. Thus, factory-matrix formation is an important, though not a sufficient function of μNS during MRV infection; μNS is multifunctional in the course of viral growth.
inclusion body; Reoviridae; silencing; siRNA; viral factory; viroplasm