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.
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.
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
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.
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.
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
Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3-Å resolution. The capsid, ~350 Å in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is in turn formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of α-helices and consistent with secondary-structure predictions for the coat protein. This is the first three-dimensional structure of a virus from the family Partitiviridae and exhibits both similarities to and differences from the so-called “T=2” capsids of other dsRNA viruses.
A low-copy component of mammalian reovirus particles is μ2, an 83-kDa protein encoded by the M1 viral genome segment and packaged within the viral core. Previous studies have identified μ2 as a nucleoside triphosphate phosphohydrolase (NTPase) as well as an RNA 5′-triphosphate phosphohydrolase (RTPase), putatively involved in reovirus RNA synthesis and/or 5′-capping. Other studies have identified μ2 as a microtubule-binding protein, which also associates with the viral factory matrix protein μNS and thereby anchors the factories to cellular microtubules during infections by most reovirus strains. To extend studies of μ2 functions during infection, we tested a small interfering RNA (siRNA) directed against the M1 plus-strand RNAs of reovirus strains Type 1 Lang (T1L) and Type 3 Dearing (T3D). The siRNA strongly suppressed μ2 expression by either strain and reduced infectious yields in a strain-dependent manner. This first strain difference was genetically mapped to the M1 genome segment and tentatively assigned to a single μ2 sequence polymorphism, Pro/Ser208, which also determines a T1L-T3D strain difference in microtubule association. The siRNA-based defect in μ2 expression was rescued by plasmids, containing silent mutations in the siRNA-targeted sequence, which encoded either T1L or T3D μ2, but the growth defect was rescued only by T1L μ2. This second strain difference was also mapped to Pro/Ser208, in that swapping this one residue between T1L and T3D μ2 reversed the rescue phenotypes. Thus, the T1L-T3D strain difference in μ2-microtubule association was correlated not only with the extent of reduction in infectious yields by the siRNA but also with the extent of rescue by plasmid-derived μ2. In addition, the rescue capacity of T1L μ2 was abrogated by nocodazole treatment, providing independent evidence for the importance of μ2-microtubule association in plasmid-based rescue. In two separate cases, the results revealed functional differences between virus- and plasmid-derived μ2. Ala substitutions within the NTP-binding motif of T1L μ2 also abrogated its rescue capacity, suggesting that the NTPase or RTPase activity of μ2 is additionally required for effective viral growth.
The 76-kDa μ1 protein of nonfusogenic mammalian reovirus is a major component of the virion outer capsid, which contains 200 μ1 trimers arranged in an incomplete T=13 lattice. In virions, μ1 is largely covered by a second major outer-capsid protein, σ3, which limits μ1 conformational mobility. In infectious subvirion particles, from which σ3 has been removed, μ1 is broadly exposed on the surface and can be promoted to rearrange into a protease-sensitive and hydrophobic conformer, leading to membrane perforation or penetration. In this study, mutants that resisted loss of infectivity upon heat inactivation (heat-resistant mutants) were selected from infectious subvirion particles of reovirus strains Type 1 Lang and Type 3 Dearing. All of the mutants were found to have mutations in μ1, and the heat-resistance phenotype was mapped to μ1 by both recoating and reassortant genetics. Heat-resistant mutants were also resistant to rearrangement to the protease-sensitive conformer of μ1, suggesting that heat inactivation is associated with μ1 rearrangement, consistent with published results. Rate constants of heat inactivation were determined, and the dependence of inactivation rate on temperature was consistent with the Arrhenius relationship. The Gibbs free energy of activation was calculated with reference to transition-state theory and was found to be correlated with the degree of heat resistance in each of the analyzed mutants. The mutations are located in upper portions of the μ1 trimer, near intersubunit contacts either within or between trimers in the viral outer capsid. We propose that the mutants stabilize the outer capsid by interfering with unwinding of the μ1 trimer.
Expression of a high-risk human papillomavirus (HPV) E7 oncoprotein is sufficient to induce aberrant centrosome duplication in primary human cells. The resulting centrosome-associated mitotic abnormalities have been linked to the development of aneuploidy. HPV type 16 (HPV16) E7 induces supernumerary centrosomes through a mechanism that is at least in part independent of the inactivation of the retinoblastoma tumor suppressor pRb and is dependent on cyclin-dependent kinase 2 activity. Here, we show that HPV16 E7 can concentrate around mitotic spindle poles and that a small pool of HPV16 E7 is associated with centrosome fractions isolated by sucrose density gradient centrifugation. The targeting of HPV16 E7 to the centrosome, however, was not sufficient for centrosome overduplication. Nonetheless, we found that HPV16 E7 can associate with the centrosomal regulator γ-tubulin and that the recruitment of γ-tubulin to the centrosome is altered in HPV16 E7-expressing cells. Since the association of HPV16 E7 with γ-tubulin is independent of pRb, p107, and p130, our results suggest that the association with γ-tubulin contributes to the pRb/p107/p130-independent ability of HPV16 E7 to subvert centrosome homeostasis.
Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein μ1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to μ1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of μ1-based thermostabilization can occur by a general mechanism of μ1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between μ1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in μ1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.
Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2CATPase, resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on mammalian orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family Reoviridae. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.
Membrane penetration by nonenveloped reoviruses is mediated by the outer-capsid protein, μ1 (76 kDa). Previous evidence has suggested that an autolytic cleavage in μ1 allows the release of its N-terminally myristoylated peptide, μ1N (4 kDa), which probably then interacts with the target-cell membrane. A substantial rearrangement of the remaining portion of μ1, μ1C (72 kDa), must also have occurred for μ1N to be released, and some regions in μ1C may make additional contacts with the membrane. We describe here a particle-free system to study conformational rearrangements of μ1. We show that removal of the protector protein σ3 is not sufficient to trigger rearrangement of free μ1 trimer and that free μ1 trimer undergoes conformational changes similar to those of particle-associated μ1 when induced by similar conditions. The μ1 rearrangements require separation of the μ1 trimer head domains but not the μ1N/C autocleavage. We have also obtained a relatively homogeneous form of the structurally rearranged μ1 (μ1*) in solution. It is an elongated monomer and retains substantial α-helix content. We have identified a protease-resistant ∼23-kDa fragment of μ1*, which contains the largely α-helical regions designated domains I and II in the conformation of μ1 prior to rearrangement. We propose that the μ1 conformational changes preceding membrane penetration or disruption during cell entry involve (i) separation of the β-barrel head domains in the μ1 trimer, (ii) autolytic cleavage at the μ1N/C junction, associated with partial unfolding of μ1C and release of μ1N, and (iii) refolding of the N-terminal helical domains of μ1C, with which μ1N was previously complexed, accompanied by dissociation of the μ1 trimer.
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.
Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein μNS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of μNS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue μNS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of μNS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of μNS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in μNS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with Aequorea victoria green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across μNS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of μNS was shown to provide a useful platform for the presentation of peptides for studies of protein-protein association through colocalization to factory-like inclusions in transfected cells.