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PubMed1986

Primary structure of leader RNA and nucleoprotein genes of the rabies genome: segmented homology with VSV.

We have determined the nucleotide sequence of the 3'region of the rabies genome (PV strain). This work is a first step in a project aimed at establishing the complete primary structure. From the 3'nucleotide sequence of the RNA genome, an octadecanucleotide complementary to the 3'extremity was constructed and used to prime cDNA synthesis. Two overlapping recombinant cDNA clones hybridizing with the nucleoprotein mRNA (NmRNA) were isolated and sequenced. The 1500 first nucleotides of the rabies genome cover two transcriptional units: the leader RNA and the NmRNA which was shown to be initiated around residue 59 by S1 nuclease protection experiments. Comparison between rabies PV and CVS strains up to residue 180 suggests a rapid evolution in the leader region. Studies of the sequence relationships between the 3'regions of two Rhabdoviruses, rabies virus and Vesicular Stomatitis Virus (VSV), demonstrate that there is a segmented homology. Stretches of highly conserved amino acids possibly involved in the interaction with the RNA genome were observed in the N protein, despite a wide divergence in the remaining sequence. In addition, the high homology between the transcription start and stop signals reflects the conservation of a similar transcriptional mechanism in these two non segmented negative strand RNA viruses.

Amino Acid Sequence↗
PubMed1995

Nucleotide and deduced amino acid sequences of the M and S genome segments of a Swedish Puumala virus isolate.

The Swedish Puumala (PUU) virus strain Vindeln 83-L20, isolated from a bank vole trapped in 1983 near Vindeln, Västerbotten county, Sweden, was characterized by nucleotide sequence analysis. The coding region of the M segment was determined by PCR followed by direct sequencing and the entire S segment was characterized by cloning and nucleotide sequence analysis. The genomic organization was found to be very similar to that of other PUU virus strains regarding open reading frames, polypeptide sizes and potential glycosylation sites. According to phylogenetic analysis 83-L20 was found to represent a new lineage within the Puumala virus serotype in the Hantavirus genus. The M segment sequence of 83-L20 was found to be more closely related to the Finnish PUU virus strains than to strains from Central Europe or from Russia. The evolutionary origin of the S segment was not as clearly resolved since the branching points of all PUU virus strains in the phylogenetic tree were nearly the same.

Amino Acid Sequence↗
PubMed2007

Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment.

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1-S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1-S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [alpha-(32)P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133-667. Within this region, a sequence stretch (aa 170-250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of 'turreted' reoviruses, these results supported the a/vxxHx(8)Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

Animals↗
PubMed1997

Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6.

A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

Bacteriophages↗
PubMed2004

Complex SNP-related sequence variation in segmental genome duplications.

There is uncertainty about the true nature of predicted single-nucleotide polymorphisms (SNPs) in segmental duplications (duplicons) and whether these markers genuinely exist at increased density as indicated in public databases. We explored these issues by genotyping 157 predicted SNPs in duplicons and control regions in normal diploid genomes and fully homozygous complete hydatidiform moles. Our data identified many true SNPs in duplicon regions and few paralogous sequence variants. Twenty-eight percent of the polymorphic duplicon sequences we tested involved multisite variation, a new type of polymorphism representing the sum of the signals from many individual duplicon copies that vary in sequence content due to duplication, deletion or gene conversion. Multisite variations can masquerade as normal SNPs when genotyped. Given that duplicons comprise at least 5% of the genome and many are yet to be annotated in the genome draft, effective strategies to identify multisite variation must be established and deployed.

Evolution, Molecular↗
PubMed1993

Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm.

Bunyamwera (BUN) virus is the prototype of the family Bunyaviridae and contains a trisegmented, single-stranded RNA genome of negative polarity. The medium (M) RNA segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, in the form of a polyprotein precursor which is cotranslationally cleaved. The gene order of the M segment is 5' G2-NSm-G1 3'. We have raised a monospecific antiserum in rabbits to a branched chain synthetic peptide to a region of the NSm protein which specifically immunoprecipitates NSm from BUN-infected cells. Indirect immunofluorescence experiments on BUN-infected cells using this antiserum gave a perinuclear staining pattern, suggesting that like the viral structural proteins, NSm localizes to the Golgi complex. An essentially full-length M segment cDNA was cloned into a recombinant vaccinia virus under control of bacteriophage T7 promoter and terminator sequences and expressed in cells co-infected with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. G1, G2, and NSm were detected in cells dually infected with the recombinant vaccina viruses, indicating that processing of the M segment-encoded precursor does not require other BUN proteins. Immunofluorescence experiments showed that the BUN glycoproteins expressed from this recombinant vaccinia virus system localized to the Golgi complex like authentic BUN proteins.

Amino Acid Sequence↗
PubMed2003

Sequence and analysis of genomic segment A and B of very virulent infectious bursal disease virus isolated from China.

A very virulent infectious bursal disease virus (vvIBDV) field strain, named SH95, was identified and characterized from flocks with vaccination failure in Shanghai. The use of random primer and a reverse transcriptase lacking RNase-H activity produced full-length cDNA copies of the viral genomic A and B segments of SH95. The 3259 base pairs (bp) of segment A and 2827 bp of segment B were amplified by long and accurate PCR in a single step, then successfully cloned and sequenced. There were five to 27 amino acid substitutions compared with other IBDV strains within the segment A polyprotein (of these, three are unique) and about nine to 38 amino acid substitutions within VP1 (of which, four are unique). The comparison of sequences encoding the polyprotein showed that vvIBDV SH95 was most closely related to Asiatic vvIBDVs, which formed a closely related group clearly distinguishable from other classical strains of IBDV. Phylogenetic analysis suggested that vvIBDV SH95 and some other Asiatic vvIBDVs were derived from similar origin. But the topology tree performed on segment B was quite different from that performed on segment A, indicating that a genetic reassortment had played an important role in the emergence of vvIBDV SH95.

Animals↗
PubMed1988

Identification and comparison of Campoletis sonorensis virus transcripts expressed from four genomic segments in the insect hosts Campoletis sonorensis and Heliothis virescens.

The Campoletis sonorensis virus (CsV; Polydnaviridae) genome consists of at least 28 closed circular superhelical (SH) DNAs. In this study we used complete clones of four SH DNAs to analyze viral transcription both in the adult parasitic wasp host Campoletis sonorensis (Ichneumonidae) and in the lepidopteran host, Heliothis virescens (Noctuidae). CsV genes are expressed in parasitized H. virescens, but no viral transcripts had been characterized from C. sonorensis until this study. The clones of the SH DNAs B, H, M, and O1 were used to probe Northern blots of poly(A)+ RNA isolated from C. sonorensis reproductive tissue and from parasitized H. virescens larvae. All four SH DNAs hybridized to viral transcripts. SH-H,-M, and -O1 hybridized to messages expressed in both hosts. SH-B and -M hybridized to transcripts that were detected only in either C. sonorensis reproductive tissue or parasitized H. virescens larvae. These results suggest that some CsV genes are expressed in a host-specific manner. In a previous study we identified a family of imperfectly conserved tandemly repeated 540-bp repeat elements on SH-B,-H and -O1 (D. A. Theilmann and M. D. Summers, 1987 J. Virol. 61; 2589-2598). Hybridization of the 540-bp repeat regions to Northern blots showed that they were all homologous to viral transcripts. A cDNA clone of a mRNA that is transcribed from the 540-bp repeat region of SH-B was isolated from a lambda gt 10 library and completely sequenced. The sequence data revealed that the 540-bp repeat element was contained within the open reading frame of this gene. These results indicate that transcribed sequences homologous to the 540-bp repeat elements represent a second gene family to be identified within the CsV genome.

Amino Acid Sequence↗
PubMed2001

Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases.

The Nodaviridae are a family of isometric RNA viruses that infect insects and fish. Their genomes, which are among the smallest known for animal viruses, consist of two co-encapsidated positive-sense RNA segments: RNA1 encodes the viral contribution to the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome, whereas RNA2 encodes the capsid protein precursor. In this study, the RNA1 sequences of two insect nodaviruses - Nodamura virus (the prototype of the genus) and Boolarra virus - are reported as well as detailed comparisons of their encoded RdRps with those of three other nodaviruses of insects and one of fish. Although the 5' and 3' untranslated regions did not reveal common features of RNA sequence or secondary structure, these divergent viruses showed similar genome organizations and encoded RdRps that had from 26 to 99% amino acid sequence identity. All six RdRp amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. A search for structural homologues in the protein structure database identified the poliovirus RdRp, 3D(pol), as the best template for homology modelling of the RNA polymerase domain of Pariacoto virus and allowed the construction of a congruent three-dimensional model. These results extend our understanding of the relationships among the RNA1 segments of nodaviruses and the predicted structures of their encoded RdRps.

3' Untranslated Regions↗
PubMed1980

Analysis of recombination in mammalian cells using SV40 genome segments having homologous overlapping termini.

Segments of SV40 DNA having homologous overlapping termini recombine to produce viable genomes in monkey cells. Frequencies of recombination on either side of a deletion marker are non-random; replication and palindromes do not appear to be essential. Since recombination involves host enzymes, a suitable system has been devised for analysing host cell recombination functions.

Animals↗
PubMed1994

Analysis of the double-stranded RNA genome segments among avian reovirus field isolates.

Nine isolates of avian reovirus (ARV) from both healthy birds and birds with different clinical illness and one commercially available vaccine strain were selected and characterized by analysis of the migration pattern of their genomic double-stranded RNA (dsRNA) segments following separation by polyacrylamide gel electrophoresis. Different electropherotypes were observed and analyzed. The results show that the dsRNA segments of ARV were markedly polymorphic among isolates within the same serotype as well as among different serotypes. The results also show no correlation between electropherotype and disease state.

Animals↗
PubMed2006

Generation of measles virus with a segmented RNA genome.

Viruses classified in the order Mononegavirales have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of Measles virus (MeV), a member of the family Paramyxoviridae in the order Mononegavirales, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3' and 5' self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.

Animals↗
PubMed2000

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions.

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Animals↗
PubMed1976

Segmented genome and nucleocapsid of La Crosse virus.

La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.

Capsid↗
PubMed1994

A member of the reoviridae (DpRV) has a ploidy-specific genomic segment in the wasp Diadromus pulchellus (Hymenoptera).

Wild and laboratory populations of the parasitoid wasp Diadromus pulchellus are infected with a new member of the reoviridae (DpRV) described in this paper. The particles of this virus possess two capsid shells (diameters: 35 and 70 nm) made up of 11 proteins. The virus is present mainly in the gut of the wasp, with smaller quantities in its venom gland. The genome of virus particles purified from haploid insects (functional males) contains 10 segments, whereas virus from diploid insects (females and sterile diploid males) contains a supernumerary 3.33-kb segment. The sequence of this dsRNA segment revealed that it is a triplicated 1050-bp motif which is 97.5% similar to the 5' region of one of the 10 basic segments, the 3.80-kb segment.

Amino Acid Sequence↗
PubMed2025

ELViS: an R package for estimating copy number levels of viral genomic segments at base-resolution.

MOTIVATION: Tumor viruses account for ∼10% of cancer diagnoses. Virally induced tumorigenesis is understood as direct signaling through oncogenes such as E6 and E7 genes in the case of human papillomavirus. Furthermore, pathogen characteristics such as viral oncogene dose may impact the disease course. To our knowledge, no tool has been proposed to assess the intra-viral copy number alterations that define the gene dose of viral oncogenes and associated suppressive pathways native to the pathogen's normal life cycle. RESULTS: We propose an R package, "ELViS," that analyzes viral copy number changes from DNA sequencing of whole viral genomes. The method adjusts for viral load with 2D transformation and segmentation to offer the relative viral gene doses. AVAILABILITY AND IMPLEMENTATION: The ELViS R package is available from https://bioconductor.org/packages/ELViS. This article used controlled access data from dbGaP (phs001713.v1.p1).

Software↗