Multiple conformations of trimeric spikes visualized on a non-enveloped virus.

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

A putative ATPase mediates RNA transcription and capping in a dsRNA virus.

mRNA transcription in dsRNA viruses is a highly regulated process but the mechanism of this regulation is not known. Here, by nucleoside triphosphatase (NTPase) assay and comparisons of six high-resolution (2.9-3.1 Å) cryo-electron microscopy structures of cytoplasmic polyhedrosis virus with bound ligands, we show that the large sub-domain of the guanylyltransferase (GTase) domain of the turret protein (TP) also has an ATP-binding site and is likely an ATPase. S-adenosyl-L-methionine (SAM) acts as a signal and binds the methylase-2 domain of TP to induce conformational change of the viral capsid, which in turn activates the putative ATPase. ATP binding/hydrolysis leads to an enlarged capsid for efficient mRNA synthesis, an open GTase domain for His217-mediated guanylyl transfer, and an open methylase-1 domain for SAM binding and methyl transfer. Taken together, our data support a role of the putative ATPase in mediating the activation of mRNA transcription and capping within the confines of the virus.

Identification of mud crab reovirus VP12 and its interaction with the voltage-dependent anion-selective channel protein of mud crab Scylla paramamosain.

Mud crab reovirus (MCRV) is the causative agent of a severe disease in cultured mud crab (Scylla paramamosain), which has caused huge economic losses in China. MCRV is a double-stranded RNA virus with 12 genomic segments. In this paper, SDS-PAGE, mass spectrometry and Western blot analyses revealed that the VP12 protein encoded by S12 gene is a structural protein of MCRV. Immune electron microscopy assay indicated that MCRV VP12 is a component of MCRV outer shell capsid. Yeast two hybrid cDNA library of mud crab was constructed and mud crab voltage-dependent anion-selective channel (mcVDAC) was obtained by MCRV VP12 screening. The full length of mcVDAC was 1180 bp with an open reading frame (ORF) of 849 bp encoding a 282 amino acid protein. The mcVDAC had a constitutive expression pattern in different tissues of mud crab. The interaction between MCRV VP12 and mcVDAC was determined by co-immunoprecipitation assay. The results of this study have provided an insight on the mechanisms of MCRV infection and the interactions between the virus and mud crab.

A newly isolated reovirus has the simplest genomic and structural organization of any reovirus.

UNLABELLED: A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events. IMPORTANCE: We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

Assembly of large icosahedral double-stranded RNA viruses.

Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.

Electron tomography reveals polyhedrin binding and existence of both empty and full cytoplasmic polyhedrosis virus particles inside infectious polyhedra.

Previous studies have described the structure of purified cytoplasmic polyhedrosis virus (CPV) and that of polyhedrin protein. However, how polyhedrin molecules embed CPV particles inside infectious polyhedra is not known. By using electron tomography, we show that CPV particles are occluded within the polyhedrin crystalline lattice with a random spatial distribution and interact with the polyhedrin protein through the A-spike rather than as previously thought through the B-spike. Furthermore, both full (with RNA) and empty (no RNA) capsids were found inside polyhedra, suggesting a spontaneous RNA encapsidating process for CPV assembly in vivo.

The functional organization of the internal components of Rice dwarf virus.

The capsid structures of particles of Rice dwarf virus that consisted of different components, namely, intact particles, empty particles lacking the 12 segments of double-stranded RNA (dsRNA), and virus-like particles composed of only the P3 core and P8 outer capsid proteins, generated with a baculovirus gene-expression system, were determined by cryo-electron microscopy. Combining the results with those of biochemical analysis, we assigned proteins of the transcriptional machinery and dsRNA to density clusters around the 5-fold axes and along the radial concentric layers, respectively. P7 protein, a component of the transcriptional machinery, was assigned to the outermost region of the density clusters. The density connecting the transcription complex to the outermost RNA densities implied interactions between the dsRNA and the P7 protein. Our structural analysis and the non-specific nucleic acid-binding activity of P7 explain the spiral organization of dsRNA around the 5-fold axis.

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

Characterization of infectious particles of grass carp reovirus by treatment with proteases.

Proteolytic cleavages play an important role in reovirus infection during entry into cells. The effects of protease digestion on the morphology, infectivity and polypeptide composition of grass carp reovirus (GCRV) were investigated. Following treatment with chymotrypsin, the different subviral particles of GCRV were isolated using density gradient centrifugation and examined by electron microscope (EM). Analysis of protein components revealed that the viral outer capsid was composed of VP5 and VP7. Of particular note, VP5 was found to primarily exist within virions as cleaved fragments, which was consistent with observations for its analogue mu1/mu1C, generated by autolysis of mu1 at the mu1N/mu1C junction for mammalian orthoreoviruses (MRVs). Meanwhile, both trypsin- and chymotrypsin-treated GCRV particles appeared to have an enhanced infectivity. Moreover, the corresponding assays between infectivity and protein component indicated that the enhancement of infectivity was correlated to the complete digestion of the outer capsid protein VP7 and partial cleavage of VP5. Overall, the results presented in this paper provided strong evidence that the proteins VP5 and VP7 of GCRV play an indispensable role in viral infection.

Three-dimensional display and arbitrary region interactive segmentation of high-resolution virus capsids from cryo-electron microscopy single particle reconstruction.

Recent advances in cryo-electron microscopy (cryo-EM) instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3-D) structure determination of virus. In order to visualize and effectively understand the 3-D structure, we present a display method based on surface rendering, which has the function of 3-D arbitrary region interactive segmentation and quantitative analysis, and integrate them into a software package called CEM-3DVDSS (cryo-EM 3-D virus display and arbitrary region segmentation system). CEM-3DVDSS consists of a complete set of modular programs for 3-D display and segmentation of icosahedral virus, which is organized under a graphical user interface and provides user-friendly options. First, we convert volume data in the MRC format obtained by cryo-EM single particle reconstruction to the format of our own software; in the preprocessing step, the original volume data are compressed and a better vector dimension is found for controlling the speed and detail of display. Then, the new volume data can be displayed and segmented using CEM-3DVDSS. We demonstrate the applicability of CEM-3DVDSS by displaying the 3-D structures of 2.5 nm (resolution) BmCPV (Bombyx mori cytoplasmic polyhedrosis virus), 2.5 nm CSBV (Chinese Sacbrood bee virus) and 1.4 nm C6/36DNV (Densonucleosis virus). As a result, both the 3-D display speed and signal-to-noise ratio of CEM-3DVDSS are improved compared with the original method, and the segmentation results become precise and more intact with additional function of quantitative analysis of 3-D structure.

Morphological and molecular characterization of a Cypovirus (Reoviridae) from the mosquito Uranotaenia sapphirina (Diptera: Culicidae).

A novel cypovirus has been isolated from the mosquito Uranotaenia sapphirina (UsCPV) and shown to cause a chronic infection confined to the cytoplasm of epithelial cells of the gastric ceca and posterior stomach. The production of large numbers of virions and inclusion bodies and their arrangement into paracrystalline arrays gives the gut of infected insects a distinctive blue iridescence. The virions, which were examined by electron microscopy, are icosahedral (55 to 65 nm in diameter) with a central core that is surrounded by a single capsid layer. They are usually packaged individually within cubic inclusion bodies (polyhedra, approximately 100 nm across), although two to eight virus particles were sometimes occluded together. The virus was experimentally transmitted per os to several mosquito species. The transmission rate was enhanced by the presence of magnesium ions but was inhibited by calcium ions. Most of the infected larvae survived to adulthood, and the adults retained the infection. Electrophoretic analysis of the UsCPV genome segments (using 1% agarose gels) generated a migration pattern (electropherotype) that is different from those of the 16 Cypovirus species already recognized. UsCPV genome segment 10 (Seg-10) showed no significant nucleotide sequence similarity to the corresponding segment of the other cypoviruses that have previously been analyzed, and it has different "conserved" termini. A BLAST search of the UsCPV deduced amino acid sequence also showed little similarity to Antheraea mylitta CPV-4 (67 of 290 [23%]) or Choristoneura fumiferana CPV-16 (33 of 111 [29%]). We conclude that UsCPV should be recognized as a member of a new Cypovirus species (Cypovirus 17, strain UsCPV-17).

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms.

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.

Electron microscopy as a reliable tool for rapid and conventional detection of enteric viral agents: a five-year experience report.

BACKGROUND AND AIM OF THE WORK: Since the introduction of the electron microscope and its subsequent development, virology has made a great step forward by the improvement of the basic knowledge on viral structure, as well as by broad application of electron microscopy (EM) to viral diagnosis. In this report, we describe a five-year experience in the use of EM for the diagnosis of enteric viral infections. METHODS: Three thousand four hundred and ninety stool specimens were analyzed at the Virology Unit (Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Italy) during a five-year period, from January 1999 to January 2004. The faecal extracts were subjected to EM after negative staining and were simultaneously cultured to evidence the presence of cytopathogenic agents. RESULTS: EM directly applied to the above specimens allowed the detection of several enteric viral agents, particularly evidencing those normally hard to cultivate (thus easily lost with culture methods). It also enabled diagnosis of dual gut infections, such as those from rotavirus and calicivirus. On the other hand, EM-based identification of viral agents after cell culture and ultracentrifugation of cytopathogenic agent-containing cellular extracts, allowed the identification of cultivable agents, such as picornaviruses, which can escape the direct EM detection if low concentrated. CONCLUSIONS: A rationalized use of EM on selected samples, such as stool, appears suitable in epidemiological or clinical conditions when a very rapid diagnosis is required to save time, including cases of suspected emerging viral infections.

An assembly model of rice dwarf virus particle.

The Phytoreovirus rice dwarf virus (RDV) has a complex nucleocapsid architecture composed of multiple proteins and RNAs. However, specific RNA-protein and protein-protein interactions involved in virion packaging have not been entirely elucidated. In order to define mechanisms governing RDV particle assembly, interactions between individual components were analyzed both in vivo and in vitro. The P7 core protein binds specifically and with high affinity to all 12 genomic RDV dsRNAs. P1, a putative RNA polymerase, P5, a putative guanyltransferase and P7 are encapsidated within the virion and also bind viral transcripts based upon in vitro binding assays. P1, P5, P7 and genomic dsRNAs were lacking in empty particles purified from infected tissues that also yielded fractions containing intact, infectious particles. In addition, P7 forms complexes with P1 and P3, a core capsid protein, in viral particles. These results indicate the possibility that core proteins and dsRNAs interact as one unit suggesting a mechanism for assortment of viral RNAs and subsequent packaging into core particles.

Molecular characterization of dsRNA segments 2 and 5 and electron microscopy of a novel reovirus from a hypovirulent isolate, W370, of the plant pathogen Rosellinia necatrix.

A hypovirulent isolate, W370, of the white root rot fungus Rosellinia necatrix has previously been shown to harbour 12 dsRNA segments. In this study, complete nucleotide sequences of segments 2 and 5 of W370 dsRNAs were determined. The nucleotide sequence of genome segment 2 was 3773 bases long with a single long open reading frame (ORF) encoding 1226 amino acid residues with a predicted molecular mass of approximately 138.5 kDa. The nucleotide sequence of segment 5 was 2089 bases long with a single long ORF, whose deduced polypeptide contained 646 amino acid residues with a predicted molecular mass of about 72 kDa. Comparative analysis showed that the deduced protein sequence of segment 2 had significant homology with the putative VP2 of Colorado tick fever virus (CTFV) and European Eyach virus (EYAV) in the genus Coltivirus, but the deduced protein sequence of segment 5 had no similarity with other virus proteins. Double-shelled spherical particles approximately 80 nm in diameter associated with W370 dsRNAs were observed in a preparation from the mycelial tissue of isolate W370. The results demonstrated that the virus associated with W370 dsRNAs is a novel reovirus of the family Reoviridae. The virus was named Rosellinia anti-rot virus (RArV).

Do viruses form lineages across different domains of life?

The scarce characterisation of the viral world has hampered our efforts to appreciate the magnitude and diversity of the viral domain. It appears that almost every species can be infected by a number of viruses. As our knowledge of viruses increases, it appears that this myriad of viruses may be organised into a reasonably low number of viral lineages including members infecting hosts belonging to different domains of life. Viruses belonging to a lineage share a common innate "self" that refers to structural and assembly principles of the virion. This hypothesis has a few consequences. All viruses are old, maybe preceding cellular life, and virus origins are polyphyletic, as opposed to the idea of a monophyletic origin of cellular life.

Co-replication of a reovirus and a polydnavirus in the ichneumonid parasitoid Hyposoter exiguae.

A recently established colony of the ichneumonid parasitoid, Hyposoter exiguae, was found to carry both a reovirus (HeRV) and a polydnavirus (HePDV). Morphogenesis of these viruses was observed in all cells comprising the ovarian calyx epithelium, apparently without detrimental effect to the parasitoid. While polydnavirus replication in H. exiguae was restricted to the calyx region, HeRV was detected in ovarioles, oviducts, midguts, malpighian tubules, and accessory glands associated with the male reproductive system. In addition, HeRV was able to infect the fat body of parasitized host larvae and to establish a persistent infection in vitro. Electron microscopy revealed that both viruses were released into the calyx fluid compartment exclusively by budding, a phenomenon rarely observed among the Reoviridae; HeRV envelopes thus obtained, however, appeared to be subsequently shed within the oviducts. HeRV particles were concentrated to near homogeneity by differential centrifugation; mature virions consisted of seven to eight structural polypeptides and 10 dsRNA genome segments. Prominent spikes were observed at the vertices of icosahedral core particles. Most, but not all, individuals comprising the H. exiguae colony appeared to be infected with HeRV, suggesting a commensal relationship between wasp and virus; however, while this association is of obvious benefit to the virus, it seems unlikely that any advantage accrues to the parasitoid which carries it.

Reovirus nonstructural protein muNS binds to core particles but does not inhibit their transcription and capping activities.

Previous studies provided evidence that nonstructural protein muNS 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 muNS. Given the possible role of muNS in particle assembly and/or transcription implied by those findings, we tested whether recombinant muNS can bind to cores in vitro. The muNS 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 muNS resulted in particle complexes of progressively decreasing buoyant density, approaching the density of protein alone when very large amounts of muNS were bound. Thus, the muNS-core interaction did not exhibit saturation or a defined stoichiometry. Negative-stain electron microscopy of the muNS-bound cores revealed that the cores were intact and linked together in large complexes by an amorphous density, which we ascribe to muNS. The muNS-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 muNS with cores greatly reduced the formation of recoated cores by stoichiometric binding of outer-capsid proteins mu1 and sigma3. These findings are consistent with the presence of muNS in transcriptase particles as described previously and suggest that, by binding to cores in the infected cell, muNS may block or delay outer-capsid assembly and allow continued transcription by these particles.

The structure of a cypovirus and the functional organization of dsRNA viruses.

Cytoplasmic polyhedrosis virus (CPV) is unique among the double-stranded RNA viruses of the family Reoviridae in having a single capsid layer. Analysis by cryo-electron microscopy allows comparison of the single shelled CPV and orthoreovirus with the high resolution crystal structure of the inner shell of the bluetongue virus (BTV) core. This suggests that the novel arrangement identified in BTV, of 120 protein subunits in a so-called 'T=2' organization, is a characteristic of the Reoviridae and allows us to delineate structural similarities and differences between two subgroups of the family--the turreted and the smooth-core viruses. This in turn suggests a coherent picture of the structural organization of many dsRNA viruses.

Preliminary characterization of a reovirus isolated from golden ide Leuciscus idus melanotus.

Some characteristics of a reovirus recently isolated from golden ide Leuciscus idus melanotus and tentatively designated as golden ide reovirus (GIRV) were determined. Spherical non-enveloped particles with an outer capsid of about 70 nm and an inner capsid of about 50 nm were observed by electron microscopy. The density of the virus determined in CsCl gradients was 1.36 g ml-1. The genome contained 11 segments of dsRNA. GIRV differed from other aquareoviruses by a slight reduction of infectivity after treatment with chloroform and by the absence of forming syncytia in cell monolayers.