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PubMed1993

Multiple double-stranded RNA segments are associated with virus particles infecting Trichomonas vaginalis.

Previous studies demonstrated that some isolates of the sexually transmitted protozoan Trichomonas vaginalis are infected with a nonsegmented, double-stranded RNA (dsRNA) virus. A reexamination of the total dsRNA extracted from several virus-harboring isolates indicated the presence of at least three dsRNAs with sizes ranging from 4.8 to 4.3 kbp. The double-stranded nature of each of the three segments was determined by hybridization experiments using riboprobes of opposite polarities obtained from cDNA generated to each of the segments. All three segments were present in agar clones originating from single organisms of T. vaginalis isolates, suggesting that the three segments were not the result of a mixed population of trichomonads harboring different sizes of dsRNA. The three segments were associated with CsCl-purified virus particles, as evidenced by electron microscopy, and RNAse treatment of the preparation containing virus particles did not destroy the dsRNAs. Finally, the individual dsRNA segments were purified for use as probes to determine whether the three dsRNAs shared any sequence homology. Each end-labeled dsRNA segment did not cross-hybridize to any of the other two segments, a finding consistent with the hybridization of labeled cDNAs to only the segments from which they were derived. These results show that the coding capacity of the dsRNA virus may be at least three times greater than that estimated earlier and illustrates further the complexity of this virus-parasite interrelationship.

Animals↗
PubMed2004

VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII.

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.

Amino Acid Sequence↗
PubMed1989

The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames.

The L-A double-stranded RNA virus of Saccharomyces cerevisiae encodes its major coat protein (80 kDa) and a minor single-stranded RNA binding protein (180 kDa) that has immunological cross-reactivity with the major coat protein. The sequence of L-A cDNA clones revealed two open reading frames (ORF), ORF1 and ORF2. These two reading frames overlap by 130 base pairs and ORF2 is in the -1 reading frame with respect to ORF1. Although the major coat protein of the viral particles is encoded by ORF1, the 180-kDa protein is derived from the entire double-stranded RNA genome by fusing ORF1 and ORF2, probably by a -1 translational frameshift. Within the overlapping region is a sequence similar to that producing a -1 frameshift by "simultaneous slippage" in retroviruses. The coding sequence of ORF2 shows a pattern characteristic of viral RNA-dependent RNA polymerases of icosahedral (+)-strand RNA viruses. Thus, the 180-kDa protein is analogous to gag-pol fusion proteins.

Amino Acid Sequence↗
PubMed2003

CPV, a stable and symmetrical machine for mRNA synthesis.

The structure of cytoplasmic polyhedrosis virus (CPV), an insect pathogen from the Reoviridae family of double-strand RNA viruses, has been determined at 8 A by electron cryomicroscopy and image reconstruction. It provides new information about the functions of these viral particles as stable machines for mRNA synthesis.

Animals↗
PubMed2006

The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.

Capsid↗
PubMed1998

Structure of double-shelled rice dwarf virus.

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-A three-dimensional structure of the 700-A-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of beta-sandwich motif and a lower domain of alpha helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.

Amino Acid Sequence↗
PubMed2001

C terminus of infectious bursal disease virus major capsid protein VP2 is involved in definition of the T number for capsid assembly.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.

Animals↗
PubMed2004

Packaging motor from double-stranded RNA bacteriophage phi12 acts as an obligatory passive conduit during transcription.

Double-stranded RNA viruses sequester their genomes within a protein shell, called the polymerase complex. Translocation of ssRNA into (packaging) and out (transcription) of the polymerase complex are essential steps in the life cycle of the dsRNA bacteriophages of the Cystoviridae family (phi6-phi14). Both processes require a viral molecular motor P4, an NTPase, which bears structural and functional similarities to hexameric helicases. In effect, switching between the packaging and the transcription mode requires the translocation direction of the P4 motor to reverse. However, the mechanism of the reversal remains elusive. Here we characterize the P4 protein from bacteriophage phi12 and exploit its purine nucleotide specificity to delineate P4 role in transcription. The results indicate that while P4 actively translocates RNA during packaging it acts as a passive conduit for RNA export. The directionality switching is accomplished via the regulation of P4 NTPase activity within the polymerase core.

Bacteriophage phi 6↗
PubMed1985

Analysis of the terminal sequences of the genome segments of four orbiviruses.

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.

Animals↗
PubMed2026

MARK1 suppresses infectious bursal disease virus replication via phosphorylating VP3.

Infectious bursal disease virus (IBDV) of the Birnaviridae family is a non-envelope, double-stranded RNA virus that encodes a VP3 protein with multiple functions, which controls viral genome replication, IFN-β production, and virus traffic in infected cells. Posttranslational modifications (PTMs), such as ubiquitination, of VP3 have been demonstrated for affecting its function and stability. To clarify the mechanism by which VP3 is regulated in IBDV infected cells, we focused on the phosphorylation of VP3. Mass spectrometry analysis identified that microtubule-affinity regulating kinases 1 (MARK1) was a kinase interacting protein of VP3. Inhibitory function of MARK1 in affecting viral replication was validated. We describe the phosphorylation event at the serine 130 (S130) and serine 163 (S163) residues of VP3 mediated by MARK1 via mass spectrometry analysis. Alanine replacement of the phosphorylation sites in VP3 significantly enhanced its RNA-binding activity. Additionally, the mutation of two serine residues led to remarkably improved in its polymerase-enhancing function. We then incorporated the two mutations to rescue recombinant IBDV. Viral growth curve analysis revealed that replication of mutant IBDV was significantly enhanced relative to wild type (WT) virus. In conclusion, we found that VP3 functions are specifically regulated by MARK1 mediated phosphorylation at S130 and S163 and that this regulation suppresses IBDV replication ultimately.

Infectious bursal disease virus↗
PubMed2025

Metagenomics reveals cryptic circulation of zoonotic viruses in Nigeria.

Zoonotic spillover events pose an ongoing threat to global health, with historic and recent viral diseases of international concern emerging from animal reservoirs 1-6. In Nigeria, limited surveillance of animal hosts at the human and animal interface continues to hinder our understanding of viruses that are cryptically circulating in animals near human dwellings with potential for consequential spillover events. We performed unbiased metagenomic next-generation sequencing (mNGS) on tissue and swab samples collected from 240 individual animals across 11 taxa (rodents, shrews, bats, goats, sheep, pigs, dogs, cats, chickens, cattle egrets, and lizards) in two Lassa-affected Nigerian states (Ondo and Ebonyi). Host-depleted sequencing reads were assembled into contigs, taxonomically classified, and subjected to phylogenetic analyses to characterize viral diversity, host associations, and evidence of cross-species transmission. Across all samples, we identified 214 distinct viral taxa spanning 33 families, of which 41% (n = 83) represent novel species by ICTV criteria. Positive-sense RNA viruses dominated (Coronaviridae, Picornaviridae, Astroviridae), followed by negative-sense RNA, single- and double-stranded DNA, and double-stranded RNA viruses. Notably, human-associated enteroviruses-including Hepatitis A virus (genotype 1b), echoviruses, coxsackieviruses, and noroviruses-were detected in goats, pigs, dogs, and chickens, indicating cryptic circulation of human pathogens in peridomestic and domesticated animals. Phylogenetic reconstructions revealed multiple cross-species viral sharing events, particularly among rodents, goats, sheep, and pigs, and extensive recombination within Nigerian Betacoronavirus 1 lineages. Interestingly we found a putative novel avian like coronavirus in rodents, goats and sheep. Ecological modelling demonstrated that host species identity, sample type, and sampling effort were primary drivers of viral richness and abundance, and that higher overall viral diversity strongly predicted cross-species transmission potential. Our integrated mNGS approach uncovered a rich and dynamic virome within animals inhabiting human-dominated environments in Nigeria, including undetected circulation of human enteric viruses. These findings underscore the importance of broad-taxonomic, real-time surveillance at human-animal interfaces to inform early-warning systems and pandemic preparedness, particularly in low-resource settings.

Journal Article↗
PubMed1995

Identification of picobirnavirus, viruses with bisegmented double stranded RNA, in rabbit faeces.

Picobirnaviruses are a novel group of viruses recently found in the faeces of several species of vertebrates. Examination by polyacrylamide gel electrophoresis of rabbit faecal samples collected in one animal facility revealed the viruses in 23 (11 per cent) of 211 samples. Further analysis by electron microscopy and caesium chloride isopycnic centrifugation confirmed the presence of picobirnaviruses in the samples. The oral inoculation of three newly weaned rabbits with purified viruses resulted in the excretion of a virus with an electropherotype similar to the inoculum, by two of the three inoculated animals. Maximal viral shedding was detected 13 days after inoculation. No sign of diarrhoea was observed either in the inoculated animals or in the virus excreting animals surveyed. No antibody activity could be detected in the paired serum samples taken from the inoculated animals.

Animals↗
PubMed1994

Structure and heterologous expression of the Ustilago maydis viral toxin KP4.

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species. In the best-characterized cases, they function by creating new pores in the cell membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double-stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double-stranded RNA but differs from other known killer toxins in several respects: it has no N-linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and it is not processed by any of the known secretory proteinases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.

Amino Acid Sequence↗
PubMed1979

Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes.

Infectious pancreatic necrosis virus of fish, infectious bursal disease virus of chickens, Tellina virus and oyster virus of bivalve molluscs, and drosophila X virus of Drosophila melanogaster are naked icosahedral viruses with an electron microscopic diameter of 58 to 60 nm. The genome of each of these viruses consists of two segments of double-stranded RNA (molecular weight range between 2.6 x 10(6) and 2.2 x 10(6), and the virion, capsid proteins fall into three size class categories (large, medium, and small; ranging from 100,000 to 27,000) as determined by polyacrylamide slab gel electrophoresis. The hydrodynamic properties of the five viruses are similar as determined by analytical ultracentrifugation and laser quasi-elastic, light-scattering spectroscopy. The calculated particle weights range between 55 x 10(6) and 81 x 10(6). Tryptic peptide comparisons of 125I-labeled virion proteins showed that five viruses are different from each other, although there was considerable overlap in the peptide maps of the three aquatic viruses, indicting a degree of relatedness. Cross-neutralization tests indicated that drosophila X, infectious pancreatic necrosis, and infectious bursal disease viruses were different from each other and from oyster and Tellina viruses. The same test showed oyster and Tellina viruses to be related. The biochemical and biophysical properties of the five viruses cannt be included in the family Reoviridae or in any of the present virus genera.

Animals↗
PubMed1992

MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly.

The MAK3 gene is necessary for propagation of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. MAK3 encodes a protein with substantial homology to the Escherichia coli rimI N-acetyltransferase that acetylates the NH2 terminus of ribosomal protein S18, and shares consensus sequences with a group of N-acetyltransferases. The NH2 terminus of the viral major coat protein encoded by L-A is normally blocked, but we find that it is unblocked in a mak3-1 mutant. L-A virus-encoded proteins produced from a cDNA clone of L-A can encapsidate the L-A (+)-strands in a wild-type host, but not in a mak3-1 mutant strain. The amount of major coat protein found in the particle fraction is reduced greater than 100-fold, and the amount in the total cell extract is reduced 5-10-fold. A modified beta-galactosidase, having as its NH2-terminal the NH2-terminal 13 residues of the L-A-encoded major coat protein, is blocked in a wild-type host, but not in a mak3-1 host. We propose that MAK3 encodes an N-acetyltransferase whose modification of the L-A major coat protein NH2 terminus is essential for viral assembly, and that unassembled coat protein is unstable.

Acetylation↗
PubMed2004

Recent trends in the molecular diagnosis of infectious bursal disease viruses.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. Two serotypes of this double-stranded RNA virus exist but only serotype 1 viruses cause disease in chickens. Detection and strain identification of IBDV is important because antigenic subtypes found within serotype 1 make it necessary to tailor vaccination programs to the antigenic type found in the bird's environment. Because conventional virus isolation and characterization are not practical for routine diagnosis of IBDV, antigen-capture enzyme-linked immunosorbent assay (ELISA) and molecular assays based on reverse transcription-polymerase chain reaction (RT-PCR) technology were developed. Compared with antigen-capture ELISA, RT-PCR assays have greater versatility and are more sensitive and specific. Strain identification has been accomplished using a variety of post-RT-PCR assays, including restriction enzyme digestion of the RT-PCR products. The resulting restriction fragment length polymorphisms (RFLP) are used to differentiate viruses into molecular groups that correlate with antigenic and pathogenic types. Recently, two types of real-time RT-PCR have been used to identify and differentiate strains of IBDV. Both methods use distance-dependent interaction between two dye molecules, known as fluorescence resonance energy transfer (FRET). The dye molecules are attached to one or more nucleotide probes that detect specific nucleotide sequences of the virus. Our laboratory has used a two-probe assay to identify single-nucleotide mutations among IBDV strains. A mutation probe is used in this assay to detect substitution mutations in a region of the viral genome that encodes a neutralizing epitope of the virus. These assays are accurate, reliable and inexpensive compared with conventional RT-PCR because they do not require RFLP or other labor-intensive post-RT-PCR assays to distinguish viral strains.

Animals↗
PubMed2006

Molecular epidemiology of human picobirnaviruses among children of a slum community in Kolkata, India.

Picobirnaviruses are a group of unclassified, non-enveloped, small spherical viruses, 35-41 nm in diameter without any apparent surface morphology. They have characteristic bisegmented double stranded RNA genome of two types namely large profile (2.3-2.6 kbp for the larger and 1.5-1.9 kbp for the smaller segment, respectively) or small profile (1.75 and 1.55 kbp for segments 1 and 2, respectively). Human picobirnaviruses (n=12 positives; 2/56 diarrhoeic children and 10/607 non-diarrhoeic children) with large (n=11) or small (n=1) genome pattern were observed in faecal specimens of children from a slum community by silver stained PAGE gels. Faecal specimen from four asymptomatic cases (P597_02_IND, K135_02_IND, A373_03_IND, A356_03_IND) and one diarrhoeic case (K135_03_IND) had genogroup I picobirnaviruses (1-CHN-97 like) showing amplicons within the 201 bp region, with primers PicoB25-PicoB43, targeting the conserved domain of RNA-dependent RNA polymerase (RdRp) gene. It was interesting to note that only the PBV strain P597_02_IND from Kolkata with large genome was closely related to a reported strain (similarity with 2-GA-91 from USA was 87% at the nucleotide level and 90% at the amino acid level). Sequence analysis showed three conserved amino acid domains as well as a highly conserved D-S-D motif, characteristic of RNA-dependent RNA polymerase gene of bisegmented, double stranded RNA viruses. Sequence data of the picobirnavirus A356_03_IND indicated strong heterogeneity with all other picobirnavirus strains sequenced till date. After nearly a decade a genogroup II picobirnavirus strain (R227_03_IND) was isolated from a diarrhoea case in the community, with small genome profile and amplified with specific primers PicoB23-PicoB24; but the sequence data showed that it was divergent from the hitherto reported prototype strain 4-GA-91 of genogroup II human picobirnaviruses.

Amino Acid Sequence↗
PubMed1996

Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

Animals↗