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The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

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.

Most dsRNA viruses have a genome-enclosing capsid that comprises 120 copies of a single coat protein (CP). These 120 CP subunits are arranged as asymmetrical dimers that surround the icosahedral fivefold axes, forming pentamers of dimers that are thought to be assembly intermediates. This scheme is violated, however, in recent structures of two dsRNA viruses, a fungal virus from family Partitiviridaeand a rabbit virus from family Picobirnaviridae, both of which have 120 CP subunits organized as dimers of quasisymmetrical dimers. In this study, we report the CP backbone trace of a second fungal partitivirus, determined in this case by electron cryomicroscopy and homology modeling. This virus also exhibits quasisymmetrical CP dimers that are connected by prominent surface arches and stabilized by domain swapping between the two CP subunits. The CP fold is dominated by α-helices, although β-strands mediate several important contacts. A dimer-of-dimers assembly intermediate is again implicated. The disordered N-terminal tail of each CP subunit protrudes into the particle interior and likely interacts with the genome during packaging and/or transcription. These results broaden our understanding of conserved and variable aspects of partitivirus structure and reflect the growing use of electron cryomicroscopy for atomic modeling of protein folds.

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein μNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase λ3, that was recruited to the structures through an association with μNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of μNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the μNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent μNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.

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.

ABSTRACTThe reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other Reoviridaefamily members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins ?1, ?2, and s2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, µ1 and s3, to produce virion-like particles. Coexpression of core shell protein ?1 and core nodule protein s2 was sufficient to yield CLPs that could withstand purification, whereas expression of ?1 alone was not, indicating a required role for s2 as a previous study also suggested. In addition, CLPs that lacked ?2 (formed from ?1 and s2 only) could not be coated with µ1 and s3, indicating a required role for ?2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of ?1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of ?1 deletion mutants, we showed that the amino-terminal 230 residues of ?1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of ?1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of ?1 with the threefold and/or fivefold copies of s2. An effective system for studies of reovirus core structure, assembly, and functions is hereby established.

ABSTRACTThe mechanisms employed by nonenveloped animal viruses to penetrate the membranes of their host cells remain enigmatic. Membrane penetration by the nonenveloped mammalian reoviruses is believed to deliver a partially uncoated, but still large (~70-nm), particle with active transcriptases for viral mRNA synthesis directly into the cytoplasm. This process is likely initiated by a particle form that resembles infectious subvirion particles (ISVPs), disassembly intermediates produced from virions by proteolytic uncoating. Consistent with that idea, ISVPs, but not virions, can induce disruption of membranes in vitro. Both activities ascribed to ISVP-like particles, membrane disruption in vitro and membrane penetration within cells, are linked to N-myristoylated outer-capsid protein µ1, present in 600 copies at the surfaces of ISVPs. To understand how µ1 fulfills its role as the reovirus penetration protein, we monitored changes in ISVPs during the permeabilization of red blood cells induced by these particles. Hemolysis was preceded by a major structural transition in ISVPs, characterized by conformational change in µ1 and elution of fibrous attachment protein s1. The altered conformer of µ1 was required for hemolysis and was markedly hydrophobic. The structural transition in ISVPs was further accompanied by derepression of genome-dependent mRNA synthesis by the particle-associated transcriptases. We propose a model for reovirus entry in which (i) primed and triggered conformational changes, analogous to those in enveloped-virus fusion proteins, generate a hydrophobic µ1 conformer capable of inserting into and disrupting cell membranes and (ii) activation of the viral particles for membrane interaction and mRNA synthesis are concurrent events. Reoviruses provide an opportune system for defining the molecular details of membrane penetration by a large nonenveloped animal virus.

ABSTRACTCells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these “viral factories” are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein µNS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3DN). In the transfected cells, the formation of µNS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller µNS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of µNS and reovirus protein µ2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing µNS and µ2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the µ2 protein from T3DNalso colocalized with µNS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing µNS and µ2 protein from T3DNand the filamentous structures containing µNS and µ2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the µ2-encoding M1 genome segment. We found that the first 40 amino acids of µNS are required for colocalization with µ2 but not for inclusion formation. Similarly, a fusion of µNS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the µ2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between µNS and µNSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of µNS. The capacity of µNS to form inclusions and to colocalize with µ2 in transfected cells suggests a key role for µNS in forming viral factories in reovirus-infected cells.

ABSTRACTEntry of mammalian reovirus virions into target cells requires proteolytic processing of surface protein s3. In the virion, s3 mostly covers the membrane-penetration protein µ1, appearing to keep it in an inactive form and to prevent it from interacting with the cellular membrane until the proper time in infection. The molecular mechanism by which s3 maintains µ1 in this inactive state and the structural changes that accompany s3 processing and µ1 activation, however, are not well understood. In this study we characterized the early steps in s3 processing and determined their effects on µ1 function and particle infectivity. We identified two regions of high protease sensitivity, “hypersensitive” regions located at residues 208 to 214 and 238 to 244, within which all proteases tested selectively cleaved s3 as an early step in processing. Further processing of s3 was required for infection, consistent with the fact that the fragments resulting from these early cleavages remained bound to the particles. Reovirus type 1 Lang (T1L), type 3 Dearing (T3D), and T1L × T3D reassortant virions differed in the sites of early s3 cleavage, with T1L s3 being cleaved mainly at residues 238 to 244 and T3D s3 being cleaved mainly at residues 208 to 214. These virions also differed in the rates at which the early cleavages occurred, with cleavage of T1L s3 occurring faster than cleavage of T3D s3. Analyses using chimeric and site-directed mutants of recombinant s3 identified carboxy-proximal residues 344, 347, and 353 as the primary determinants of these strain differences. The spatial relationships between these more carboxy-proximal residues and the hypersensitive regions were discerned from the s3 crystal structure. The results indicate that proteolytic processing of s3 during reovirus disassembly is a multistep pathway with a number of molecular determinants.

Cells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or "factories," where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L x T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the mu2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the mu2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of alpha-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of mu2 with the changes Pro-208 to Ser in a background of T1L mu2 and Ser-208 to Pro in a background of T3D mu2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the mu2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.

ABSTRACTCells infected with mammalian reoviruses often contain large perinuclear inclusion bodies, or “factories,” where viral replication and assembly are thought to occur. Here, we report a viral strain difference in the morphology of these inclusions: filamentous inclusions formed in cells infected with reovirus type 1 Lang (T1L), whereas globular inclusions formed in cells infected with our laboratory's isolate of reovirus type 3 Dearing (T3D). Examination by immunofluorescence microscopy revealed the filamentous inclusions to be colinear with microtubules (MTs). The filamentous distribution was dependent on an intact MT network, as depolymerization of MTs early after infection caused globular inclusions to form. The inclusion phenotypes of T1L × T3D reassortant viruses identified the viral M1 genome segment as the primary genetic determinant of the strain difference in inclusion morphology. Filamentous inclusions were seen with 21 of 22 other reovirus strains, including an isolate of T3D obtained from another laboratory. When the µ2 proteins derived from T1L and the other laboratory's T3D isolate were expressed after transfection of their cloned M1 genes, they associated with filamentous structures that colocalized with MTs, whereas the µ2 protein derived from our laboratory's T3D isolate did not. MTs were stabilized in cells infected with the viruses that induced filamentous inclusions and after transfection with the M1 genes derived from those viruses. Evidence for MT stabilization included bundling and hyperacetylation of a-tubulin, changes characteristically seen when MT-associated proteins (MAPs) are overexpressed. Sequencing of the M1 segments from the different T1L and T3D isolates revealed that a single-amino-acid difference at position 208 correlated with the inclusion morphology. Two mutant forms of µ2 with the changes Pro-208 to Ser in a background of T1L µ2 and Ser-208 to Pro in a background of T3D µ2 had MT association phenotypes opposite to those of the respective wild-type proteins. We conclude that the µ2 protein of most reovirus strains is a viral MAP and that it plays a key role in the formation and structural organization of reovirus inclusion bodies.

ABSTRACTKinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein µ1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound µ1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of µ1 as a determinant of thermostability. Intact virions, which contain ?3 bound to µ1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for ?3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.

The reovirus L2 genome segment encodes the core spike protein λ2, which mediates enzymatic reactions in 5′ capping of the viral plus-strand transcripts. Complete nucleotide-sequence determinations were made for the L2 genome segments of eight mammalian reoviruses, including the prototype isolates of serotypes 1 and 2: Lang (T1L) and Jones (T2J), respectively. Each L2 segment was found to be 3912 or 3915 bases in length. Partial nucleotide-sequence determinations were also made for the 3916-base L2 segment of reovirus type 3 Dearing (T3D), the prototype isolate of serotype 3. The whole-genome sequence of reovirus T3D was reported previously. The T1L L2 analysis represents completion of the whole-genome sequence of that isolate as well. The T2J L2 analysis leaves only the sequence of the M1 segment yet to be reported from the genome of that isolate. The T2J M1 sequence made available from analysis in another lab was used for initiating whole-genome comparisons of reoviruses T1L, T2J, and T3D in this report. The nine L2 gene sequences and deduced λ2 protein sequences were used to gain further insights into the biological variability, structure, and functions of λ2 through comparisons of the sequences and reference to the crystal structure of core-bound λ2. Phylogenetic comparisons suggest the presence of three evolutionary lines of divergent L2 alleles among the nine isolates. Localized regions of conserved amino acids in the λ2 crystal structure include active-site clefts of the RNA capping enzyme domains, sites of interactions between λ2 domains within the pentameric spike structure, and sites of interaction between λ2 subunits and other proteins in viral particles.

ABSTRACTMammalian reoviruses, prototype members of theReoviridaefamily of nonenveloped double-stranded RNA viruses, use at least three proteins—?1, µ1, and ?3—to enter host cells. ?1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ?1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ?1, µ1, and ?3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ?1. The recoated particles bound to and infected cultured cells in a ?1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ?1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ?1 protein. Additional experiments showed that recoated particles containing ?1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ?1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ?1 with respect to its structure, assembly into particles, and roles in entry.

The particle-associated reovirus polymerase synthesizes mRNA within only certain viral particle types. Reovirus cores, subviral particles lacking outer capsid proteins μ1, ς3, and ς1, produce mRNA and abortive transcripts. Reovirus virions, which contain complete outer capsids, cannot produce mRNA and produce few abortive transcripts. Recoated cores are virion-like particles generated by the addition of recombinant outer capsid proteins to cores. We used recoated cores to analyze transcriptional regulation by reovirus outer capsid proteins. Partially recoated particles, containing less than virion amounts of μ1 and ς3, synthesized mRNA at levels inversely proportional to outer capsid protein levels. Fully recoated cores exhibited undetectable mRNA synthesis levels, as did virions. However, recoated cores produced high levels of abortive transcripts. Recoated core abortive transcripts remained particle-associated and appeared to inhibit further abortive transcript production. Proteolysis of recoated cores removing μ1 and ς3 released accumulated abortive transcripts and relieved inhibition of mRNA and abortive transcript synthesis. These results suggest transcriptional elongation, but not initiation, is blocked by virion-like amounts of μ1 and ς3. Particle-associated abortive transcripts may down-regulate transcriptional initiation. Minor outer capsid protein ς1 had no demonstrable effect on transcriptional activities. Transcriptional regulation may ensure progeny virions do not compete with transcribing particles for ribonucleoside triphosphates.

ABSTRACTReovirus nonstructural protein ?NS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of ?NS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of ?NS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The ?NS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, ?NS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, ?NS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl2, distinguishing it from a classical ATP-dependent helicase. These properties of ?NS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for ?NS in replication of the reovirus RNA genome.

ABSTRACTPrevious studies provided evidence that nonstructural protein µNS of mammalian reoviruses is present in particle assembly intermediates isolated from infected cells. Morgan and Zweerink (Virology 68:455–466, 1975) showed that a subset of these intermediates, which can synthesize the viral plus strand RNA transcripts in vitro, comprise core-like particles plus large amounts of µNS. Given the possible role of µNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant µNS can bind to cores in vitro. The µNS protein bound to cores, but not to two particle forms, virions and intermediate subvirion particles, that contain additional outer-capsid proteins. Incubating cores with increasing amounts of µNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of µNS were bound. Thus, the µNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the µNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to µNS. The µNS-core complexes retained the capacity to synthesize the viral plus strand transcripts as well as the capacity to add methylated caps to the 5' ends of the transcripts. In vitro competition assays showed that mixing µNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins µ1 and ?3. These findings are consistent with the presence of µNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, µNS may block or delay outer-capsid assembly and allow continued transcription by these particles.

The 144-kDa λ2 protein of mammalian reovirus catalyzes a number of enzymatic activities in the capping of reovirus mRNA, including the transfer of GMP from GTP to the 5′ end of the 5′-diphosphorylated nascent transcript. This reaction proceeds through a covalently autoguanylylated λ2-GMP intermediate. The smaller size of RNA capping guanylyltransferases from other organisms suggested that the λ2-associated guanylyltransferase would be only a part of this protein. Limited proteinase K digestion of baculovirus-expressed λ2 was used to generate an amino-terminal Mr42,000 fragment that appears to be both necessary and sufficient for guanylyltransferase activity. Although lysine 226 was identified by previous biochemical studies as the active-site residue that forms a phosphoamide bond with GMP in autoguanylylated λ2, mutation of lysine 226 to alanine caused only a partial reduction in guanylyltransferase activity at the autoguanylylation step. Alanine substitution for other lysines within the amino-terminal region of λ2 identified lysine 190 as necessary for autoguanylylation and lysine 171 as an important contributor to autoguanylylation. A novel active-site motif is proposed for the RNA guanylyltransferases of mammalian reoviruses and otherReoviridaemembers.

Rotavirus open cores prepared from purified virions consist of three proteins: the RNA-dependent RNA polymerase, VP1; the core shell protein, VP2; and the guanylyltransferase, VP3. In addition to RNA polymerase activity, open cores have been shown to contain a nonspecific guanylyltransferase activity that caps viral and nonviral RNAs in vitro.In this study, we examined the structure of RNA caps made by open cores and have analyzed open cores for other capping-related enzymatic activities. Utilizing RNase digestion and thin-layer chromatography, we found that the majority (∼70%) of caps made by open cores contain the tetraphosphate linkage, GppppG, rather than the triphosphate linkage, GpppG, found on mRNAs made by rotavirus double-layered particles. Enzymatic analysis indicated that the GppppG caps resulted from the lack of a functional RNA 5′-triphosphatase in open cores, to remove the γ-phosphate from the RNA prior to capping. RNA 5′-triphosphatases commonly exhibit an associated nucleoside triphosphatase activity, and this too was not detected in open cores. Caps of some RNAs contained an extra GMP moiety (underlined) and had the structure 3′-GpGp(p)ppGpGpC-RNA-3′. The origin of the extra GMP is not known but may reflect the cap serving as a primer for RNA synthesis. Methylated caps were produced in the presence of the substrate, S-adenosyl-l-methionine (SAM), indicating that open cores contain methyltransferase activity. UV cross-linking showed that VP3 specifically binds SAM. Combined with the results of earlier studies, our results suggest that the viral guanylyltransferase and methyltransferase are both components of VP3 and, therefore, that VP3 is a multifunctional capping enzyme.

To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5′ triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein λ1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the λ1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced λ1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The λ1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the λ1 open reading frame to within 60 nucleotides of the plus-strand 3′ end for T3D and the other isolates sequenced, in keeping with the short 3′ nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D λ1 were located within the amino-terminal 500 residues of λ1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and λ1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of λ1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in λ1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure–function relationships for the λ1 core protein.

Nucleotide sequences of the mammalian orthoreovirus (reovirus) type 1 Lang and type 2 Jones M3 gene segments were newly determined. The nucleotide sequence of the reovirus type 3 Dearing M3 segment also was determined to compare with a previously reported M3 sequence for that isolate. Comparisons showed Lang and Dearing M3 to be more closely related than either was to Jones M3, consistent with previous findings for other reovirus gene segments. The μNS protein sequences deduced from each M3 segment were shown to be related in a similar pattern as the respective nucleotide sequences and to contain several regions of greater or less than average variability among the three isolates. Identification of conserved methionine codons near the 5′ ends of the Lang, Jones, and Dearing M3 plus strands lent support to the hypothesis that μNSC, a smaller protein also encoded by M3, arises by translation initiation from a downstream methionine codon within the same open reading frame as μNS. Other analyses of the deduced protein sequences indicated that regions within the carboxyl-terminal third of μNS and μNSC from each isolate have a propensity to form α-helical coiled coils, most likely coiled-coil dimers. The new sequences will augment further studies on μNS and μNSC structure and function.

ABSTRACTReovirus outer-capsid proteins µ1, ?3, and ?1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed µ1 and ?3 onto purified cores that lack µ1, ?3, and ?1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein ?1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of µ1 and ?3 onto cores had induced rearrangement of the pentameric ?2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the ?1 protein. To examine the utility of r-cores for genetic dissections of µ1 functions in reovirus entry, we generated r-cores containing a mutant form of µ1 that had been engineered to resist cleavage at the d:f junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in d:f cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.

ABSTRACTStructure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein ?3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed ?3 protein to infectious subvirion particles (ISVPs) that lack ?3. A level of ?3 binding approaching 100% of that in native virions was routinely achieved. The ?3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study ?3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of ?3 but contain outer-capsid protein µ1/µ1C mostly cleaved at the d-f junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that ?3, and not the d-f cleavage of µ1/µ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric ?3 proteins to localize the primary determinants of a strain-dependent difference in ?3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.

Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an essential step in productive infection. Proteolytic processing takes the form of a series of cleavages of outer-capsid proteins ς3 and μ1/μ1C. Previous studies showed an effect of both NH4Cl and E-64 on these cleavages, indicating that one or more of the acid-dependent cysteine proteinases in mammalian cells (cathepsins B and L, for example) is required; however, these studies did not address whether acid-dependent aspartic proteinases in those cells (cathepsin D, for example) may also be required. To determine the role of aspartic proteinases in reovirus entry, studies with pepstatin A, a specific inhibitor of aspartic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Experiments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibited within the tested range of pepstatin A concentrations bothin vitroand within living cells. In other experiments, virion-bound ς3 protein was shown to be a poor substrate for cleavage by cathepsin Din vitro,consistent with the findings with inhibitors. In sum, the data indicate that cathepsin-D-like aspartic proteinases provide little or no activity toward proteolytic events required for infection of L or MDCK cells with reovirus virions.

Structures of biological macromolecules determined by transmission cryoelectron microscopy (cryo-TEM) and three-dimensional image reconstruction are often displayed as surface-shaded representations with depth cueing along the viewed direction (Z cueing) Depth cueing to indicate distance from the center of virus particles (radial-depth cueing, or R cueing) has also been used. We have found that a style of R cueing in which color is applied in smooth or discontinuous gradients using the IRIS Explorer software is an informative technique for displaying the structures of virus particles solved by cryo-TEM and image reconstruction. To develop and test these methods, we used existing cryo-TEM reconstructions of mammalian reovirus particles. The newly applied visualization techniques allowed us to discern several new structural features, including sites in the inner capsid through which the viral mRNAs may be extruded after they are synthesized by the reovirus transcriptase complexes. To demonstrate the broad utility of the methods, we also applied them to cryo-TEM reconstructions of human rhinovirus, native and swollen forms of cowpea chlorotic mottle virus, truncated core of pyruvate dehydrogenase complex fromSaccharomyces cerevisiae,and flagellar filament ofSalmonella typhimurium.We conclude that R cueing with color gradients is a useful tool for displaying virus particles and other macromolecules analyzed by cryo-TEM and image reconstruction.

We used low-temperature, high-resolution scanning electron microscopy (cryo-HRSEM) to visualize surface structures on individual reovirus particles. Both intact virions and two forms of subvirion particles—infectious subvirion particles and cores—were examined, and despite some distortion of particles during specimen preparation and viewing in the microscope, the images obtained by cryo-HRSEM exhibited a level of interpretable detail not routinely achieved by other methods without image averaging. Cryo-HRSEM images of discrete reovirus particles were used to characterize and confirm features of the outer protein capsid of this virus by comparison with image reconstructions previously derived from cryotransmission electron microscopy. Distinct surface features attributable to each of the four outer-capsid proteins were identified. In addition, cryo-HRSEM images confirmed that significant changes occur on the surfaces of individual reovirus particles during disassembly and entry of cells and that the reovirus outer capsid is organized as a left-handed T=13 icosahedron. Several unique capabilities and potential uses suggest that cryo-HRSEM has a place alongside other, more established methods for molecular characterizations of virus particles.

ABSTRACTA requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein ?1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes ?1 cleavage upon conversion of virions to ISVPs. We examined the ?1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and ?1 cleavage. The ?1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the ?1 proteins of the remaining strains. Thr249occupies the dposition of a heptad repeat motif predicted to stabilize ?1 oligomers through a-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of ?1 known as the neck. Substitution of Thr249with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D ?1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the ?1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves ?1 after Arg245. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of ?1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for ?1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.

ABSTRACTMammalian reovirus virions undergo partial disassembly of the outer capsid upon exposure to proteases in vitro, producing infectious subvirion particles (ISVPs) that lack protein ?3 and contain protein µ1/µ1C as endoprotease-generated fragments µ1d/d and f. ISVPs are thought to be required for two early steps in reovirus infection: membrane penetration and activation of the particle-bound viral transcriptase complexes. Genetic and biochemical evidence implicates outer-capsid protein µ1 in both these steps. To determine whether the cleavage of µ1/µ1C is relevant to the unique properties of ISVPs, we analyzed the properties of novel subvirion particles that lacked ?3 yet retained µ1/µ1C in an uncleaved but cleavable form. These detergent-plus-protease subvirion particles (dpSVPs) were produced by treating virions with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents. Infections with dpSVPs in murine L or canine MDCK cells provided evidence that the cleavage of µ1/µ1C during viral entry into these cells is dispensable for reovirus infection. Additionally, dpSVPs behaved like ISVPs in their capacity to permeabilize lipid bilayers and to undergo transcriptase activation in vitro, supporting the conclusion that cleavage of µ1/µ1C to µ1d/d and f during viral entry is not required for either membrane penetration or transcriptase activation in cells. The capacity of alkyl sulfate detergents to inhibit the cleavage of µ1/µ1C in a reversible fashion suggests a specific association between virus particle and detergent micelles that may mimic virus particle-phospholipid membrane interactions during reovirus entry into cells.

Reovirus nonstructural protein ςNS exhibits a ssRNA-binding activity thought to be involved in assembling the reovirus mRNAs for genome replication and virion morphogenesis. To extend analysis of this activity, recombinant ςNS (rςNS) was expressed in insect cells using a recombinant baculovirus. In infected-cell extracts, rςNS was found in large complexes (≥30 S) that were disassembled into smaller, 13–19 S complexes upon treatment with RNase A. RςNS also bound to poly(A)–Sepharose beads both before and after purification. Treatment with high salt during purification caused rςNS to sediment in even smaller, 7–9 S complexes, consistent with more complete loss of RNA. To localize the RNA-binding site, limited proteolysis was used to fragment the rςNS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amino terminus of rςNS, and the resulting protein no longer bound to poly(A). In addition, when rςNS in cell extracts was treated with thermolysin to generate the amino-terminally truncated form, it sedimented at 7–9 S, also consistent with the loss of RNA-binding capacity. To confirm these findings, a deletion mutant lacking amino acids 2–11 was constructed and expressed in insect cells from a recombinant baculovirus. The mutant protein in cell extracts showed greatly reduced poly(A)-binding activity and sedimented as 7–9 S complexes. These data suggest that the first 11 amino acids of ςNS, which are predicted to form an amphipathic α-helix, are important for both ssRNA binding and formation of complexes larger than 7–9 S.

One or more proteins in mammalian reovirus core particles mediate two RNA methylation activities, (guanosine-7-N)-methyltransferase and (guanosine-2′-O)-methyltransferase, that contribute to forming the 5′ cap 1 structure on viral mRNA. We used UV irradiation to identify core proteins that bindS-adenosyl-l-methionine (SAM), the methyl-group donor for both methyltransferases. A [methyl-3H]SAM-binding site was observed among the reovirus λ proteins; was shown to be specific by competition with low levels of S-adenosyl-l-homocysteine, the product of methyl-group transfer from SAM; and was subsequently localized to protein λ2. λ2 mediates the guanylyltransferase reaction in cap formation and was previously proposed to mediate one or both methylation reactions as well. SAM binding was demonstrated for both λ2 in cores and λ2 expressed in insect cells from a recombinant baculovirus. Using three different methods to cleave λ2, a binding site for SAM was tentatively localized to a central region of λ2, between residues 792 and 1100, which includes a smaller region with sequence similarity to the SAM-binding pocket of other methyltransferases. Alanine substitutions at positions 827 and 829 within this predicted binding region greatly reduced the capacity of baculovirus-expressed λ2 protein to undergo UV cross-linking to SAM but had no effects on either the guanylyltransferase activity of this protein or its conformation as judged by partial proteolysis, suggesting that one or both of these residues is essential for SAM binding. Based on these findings, we propose that the two methyltransferase activities involved in mRNA capping by reovirus cores utilize a single SAM-binding pocket within a central region of λ2.

The structure of mammalian orthoreovirus top component particles, which are profoundly deficient in the content of double-stranded RNA genome, was determined at 30 Å resolution by transmission cryoelectron microscopy and three-dimensional image reconstruction. Previously undetected, ordered densities, appearing primarily as pentameric flowers in the reconstruction, were seen to extend 65 Å inwardly from the inner capsid at the icosahedral fivefold axes. Identically positioned but lower density elements were observed in two types of partially uncoated top component particles obtained by limited proteolysis. The levels of three inner-capsid proteins—λ1, λ3, and μ2—were reduced in concert with the internal densities during proteolytic uncoating. Since λ3 contains the catalytic regions of the viral RNA polymerase and since both λ1 and μ2 appear to play roles in transcription or mRNA capping, the internal structures are concluded to be complexes of the viral transcriptase-related enzymes. The findings have implications for the mechanisms of transcription and mRNA capping by orthoreovirus particles.

ABSTRACTThe flagellated protozoan Trichomonas vaginalisis an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalisare 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 vaginalisvirus 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 Totiviridaefamily 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. vaginalisor 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 Totiviridaefamily 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.IMPORTANCETrichomonas vaginaliscauses ~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.