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PubMed2001

Ribozymes that cleave reovirus genome segment S1 also protect cells from pathogenesis caused by reovirus infection.

Reovirus genome segment S1 encodes protein final sigma1, which is the receptor binding protein, modulates tissue tropism, and specifies the nature of the antiviral immune response. It makes up less than 2% of reovirus particles and is synthesized in very small amounts in infected cells. Any antiviral strategy aimed at reducing specifically the expression of this genome segment should, in principle, reduce the infectivity of the virus. To test this hypothesis, we have assembled two hammer-head motif-containing ribozymes (Rzs) targeted to cleave at the conserved B and C domains of the reovirus s1 RNA. Protein-independent but Mg(2+)-dependent sequence-specific cleavage of s1 RNA was achieved by both the Rzs in trans. Cells that transiently express these Rzs, when challenged with reovirus, were protected against the cytopathic effects caused by the virus. This protection correlated with the specific intracellular reduction of s1 transcripts that was due to their cleavage by the Rzs. Rz-treated cells that were challenged with reovirus showed almost complete disappearance of protein final sigma1 without significantly altering the levels of the other reovirus structural proteins. Thus, Rzs, besides acting as antiviral agents, could be exploited as biological tools to delineate specific functions of target genes.

Animals↗

PubMed1992

Detection of bluetongue virus using a cDNA probe derived from genome segment 4 of bluetongue virus serotype 2.

The double-stranded (ds) RNA genome segment 4 of bluetongue virus (BTV) serotype 2 was cloned and used as a serogroup-specific complementary (c) DNA probe for BTV diagnosis. A cDNA representing a 60% copy of genome segment 4 BTV-2 prototype was produced. The specificity of the cDNA probe was determined by hybridizing this probe to a northern blot of dsRNA (separated by polyacrylamide gel electrophoresis) of plaque-purified BTV-2 prototype. This cDNA probe was then used to hybridize to the RNA samples. Because the probe hybridized to all BTV samples but not to epizootic hemorrhagic disease virus samples, it appears to be a group-specific probe that could be used in BTV diagnosis.

Animals↗

PubMed1987

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups A, C, F, and G to genome segments.

Crosses were performed between prototype temperature-sensitive (ts) mutants of simian rotavirus SA11 representing reassortment groups A, C, F, and G and ts mutants of rhesus rotavirus RRV that belonged to different reassortment groups. Wild-type (ts+) reassortant progeny were identified by plaque formation at nonpermissive temperature (39 degrees), picked, and grown to high titer. The ts+ phenotype of the resulting progeny clones was verified by titration at 39 degrees and 31 degrees. The electropherotypes of the ts+ clones were determined by electrophoresis, and parental origin of each genome segment was assigned by comparison of segment mobility to parental markers. Analysis of the parental origin of genome segments in the ts+ reassortants derived from SA11 ts X RRV ts crosses revealed the following map locations of the SA11 prototype ts mutants: tsA(778), segment 4; tsC(606), segment 1; tsF(2124), segment 2; and tsG(2130), segment 6. The assignment of tsA was made on the basis of genome segment segregation in two independent crosses with each of two independent RRV ts mutants. The assignment of tsC was made on the basis of segregation in only a single cross with an RRV ts mutant; however, a larger number of progeny clones were examined from this cross. The lesion of tsF was mapped with data from three independent crosses using two different RRV ts mutants. The assignment of tsG was made on the basis of segregation in three independent crosses, two with RRV ts mutants and one with Wa. The assignments of tsA, tsC, and tsF were confirmed in crosses between RRV ts mutants representing those reassortment groups, and SA11 ts mutants in other reassortment groups.

Chromosome Mapping↗

PubMed1989

Biological and immunological characterization of a simian rotavirus SA11 variant with an altered genome segment 4.

We have studied a variant virus isolated from a stock of SA11 virus (H. G. Pereira, R. S. Azeredo, A. M. Fialho, and M. N. P. Vidal, 1984, J. Gen. Virol. 65, 815-818). This virus, designated 4F, was initially identified by its faster electrophoretic mobility for genome segment 4. The variant was analyzed to determine if the altered electrophoretic mobility of genome segment 4 could be correlated with phenotypic changes. Comparison of our standard laboratory SA11 virus (clone 3) with the 4F variant showed the following: (i) The 4F variant possesses a viral hemagglutinin (VP4) with a higher apparent molecular weight than clone 3. (ii) The 4F variant produces large plaques when assayed in vitro, as compared to clone 3. (iii) The 4F variant produces plaques in the absence of proteolytic enzymes, whereas clone 3 does not. (iv) The 4F variant reacts with serotype-specific neutralizing monoclonal antibodies to VP7, but fails to react with several neutralizing anti-VP4 monoclonal antibodies generated to SA11 clone 3. (v) The 4F variant grows to a higher titer and is more stable than clone 3. (vi) The 4F variant produces a VP4 that appears to be more susceptible to cleavage by trypsin than is the VP4 of clone 3. Further analyses with the 4F variant may lead to an understanding of the molecular basis for these altered phenotypes that appear to be related, at least in part, to the product of genome segment 4.

Antigens, Viral↗

PubMed1986

A new method for extracting circular and supercoiled genome segments from cytoplasmic polyhedrosis virus.

The genome of a cytoplasmic polyhedrosis virus (CPV) consisting of 10 segments was extracted by a new two-step extraction method. The results from polyacrylamide gel electrophoresis, phenol and diepoxybutane treatment and electron microscopic examination indicated the extracted genome segments to have circular and supercoiled structures as genome-protein(s) complexes. Multiple cycles of the transcription of each segmented genome of CPV may take place on the circular structure of the segment.

Animals↗

PubMed1981

A sensitive method for the production of diagnostic fingerprints of the genome segments of field isolates of rotavirus.

The 3'-terminal labelling procedure for analyzing the genome segment profile of field isolates of rotavirus (Clarke and McCrae, 1981, J. Virol. Methods 2, 203) has been further developed to produce a rapid and sensitive method for generating diagnostic fingerprints from individual species of double-stranded RNA. The fingerprints were obtained by a one-dimensional resolution of overlapping terminally labelled oligonucleotides produced by partial nuclease digestion with a base-specific nuclease. This fingerprinting method should be of great value in characterizing the molecular details of genome segment variation and will facilitate detailed molecular epidemiological studies of this important virus group.

Animals↗

PubMed1984

Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode.

The purified genomic dsRNA of bluetongue virus type 1 (BTV 1) was separated into 10 size classes by polyacrylamide gel electrophoresis. These genome segments were recovered individually from the gel, denatured with methylmercuric hydroxide and translated in vitro in a rabbit reticulocyte lysate system. The virus-specific proteins synthesised in vitro were compared by polyacrylamide gel electrophoresis with proteins from purified virus particles and cores and with virus-specified proteins synthesized in BHK 21 cells (in vivo). The identify of those BTV 1 proteins synthesized in vivo and in vitro which showed similar electrophoretic mobilities, was confirmed by electrophoretic analysis of their partial protease digests. The results of these experiments have allowed the assignment of each of the genome segments of BTV 1 to the protein(s) which it encodes and consequently to the structural and nonstructural proteins found in the infected cell.

Animals↗

PubMed2005

The goose reovirus genome segment encoding the minor outer capsid protein, sigma1/sigmaC, is bicistronic and shares structural similarities with its counterpart in Muscovy duck reovirus.

Reoviruses have recently been shown to be associated with disease in young geese and to be involved in epizooties of severe outcome in Hungary. To assess the genetic variability among these pathogenic goose reoviruses (GRVs), we sequenced the S4 genome segment of five GRV strains isolated from different diseased flocks. We found that the GRV S4 genome segment, consisting of two partially overlapping open reading frames (ORFs), shares substantial structural similarity with its counterpart in muscovy duck reoviruses (DRVs). ORF1 is predicted to encode a polypeptide highly similar to the p10 polypeptide of DRV, and ORF2 supposedly encodes the minor outer capsid protein, sigma1/sigmaC. In one of the five GRV strains examined, we identified a single uracil base insertion close to the middle of ORF2. This insertion resulted in a frameshift and in concomitant acquisition of a termination codon (UAA) a few codons downstream, apparently causing truncation of the C-terminal part of the protein. The functional consequences of this assumed mutation, which would result in loss of more than a half of the protein, have yet to be determined. Nonetheless, the sequence and structural similarities between the genome segment encoding sigmal/sigmaC in GRVs and DRVs suggest that these viruses belong to a species distinct from other established species within subgroup 2 of orthoreoviruses.

Amino Acid Sequence↗

PubMed1984

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2.

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.

Amino Acid Sequence↗

PubMed1995

Packaging of multiple copies of reduced-size genomic segments by bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. Packaging of individual segments is dependent upon unique packaging sequences of about 300 nucleotides near the 5' ends of the segments. We have prepared segments L, M, and S with internal deletions that decrease their size by as much as sixfold without affecting either their packaging sequences or their 3' ends. Although packaging of genomic segments is normally very precise, with only one of each in a procapsid, these smaller segments are packaged in multiples such that the total number of nucleotides for each segment class approaches that of the normal genomic segment.

Bacteriophages↗

PubMed2001

Characterization of the infectious salmon anemia virus genomic segment that encodes the putative hemagglutinin.

The genomic segment encoding the putative hemagglutinin of infectious salmon anemia virus (ISAV) is described. Expression of the putative hemagglutinin in a salmon cell line demonstrated hemadsorptive properties of the protein for salmon erythrocytes. The polypeptide was recognized by an ISAV-specific monoclonal antibody. Nucleotide sequencing indicated the occurrence of a variable region in the hemagglutinin gene.

Amino Acid Sequence↗

PubMed1989

Structure of rearranged genome segment 11 in two different rotavirus strains generated by a similar mechanism.

The structures of the rearranged genomic segment 11 of two spontaneous swine rotavirus strains were determined. We found that the rearrangements involved the duplication of normal segment 11 in a head-to-tail orientation, and partial deletions in both monomers. The open reading frame for VP11, the protein encoded by normal segment 11, was maintained. We also showed that the two rearranged genes were transcribed into RNA molecules of the same length as their corresponding genomic segments.

Animals↗

PubMed1996

Genomic rearrangement in genome segment 12 of rice dwarf phytoreovirus.

The genome segment 12 (S12) of rice dwarf phytoreovirus (RDV) isolated from the Philippines (RDV-P) and of a variant (RDV-S-6) of RDV severe strain (RDV-S) migrated abnormally slower during polyacrylamide gel electrophoresis than that of the isolate maintained at Hokkaido University (RDV-H). Nucleotide sequence analysis revealed that rearrangement had occurred in these segments, affecting the open reading frame. A polypeptide encoded by S12 (Pns12) of RDV-P had a duplication of 28 amino acids while 1/3 of the carboxyl terminus of Pns12 was deleted in RDV-S-6 by premature termination due to a frameshift. RDV-S is always present in plants infected with the RDV-S-6 variant, suggesting that Pns12 of RDV-S-6 is defective. On the other hand, Pns12 of RDV-P was expressed and appeared to be functional in infected cells in spite of the duplication, as demonstrated by immunoblot analyses using antibody raised against Pns12 expressed in Escherichia coli.

Base Sequence↗

PubMed2004

Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus.

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

Animals↗

PubMed1991

Expression of rotavirus proteins encoded by alternative open reading frames of genome segment 11.

The nucleotide sequence of rotavirus genome segment 11 shows that this gene contains three potential open reading frames. We used several approaches to determine whether any polypeptides other than NS26, the primary protein product, are expressed. In particular, we sought to determine whether the strong out-of-phase start codon present at nucleotides 80-82, which would encode a protein of 92 amino acids, is used in vivo or in cell-free systems. Several modifications of gene 11 were made and found to produce proteins from the different initiation codons in cell-free transcription-translation systems. The protein from the out-of-phase open reading frame was shown to be expressed in rotavirus-infected MA104 cells; this was demonstrated using monospecific sera prepared to this protein expressed in Spodoptera frugiperda insect cells infected with a baculovirus recombinant containing only the out-of-phase open reading frame. The origin of some of the lower-molecular-weight bands serologically related to the primary product of gene 11, NS26, was also studied by selective immunoprecipitation using two different sera made from recombinant baculovirus lysates. All of these polypeptides are present in infected cells in a complex which is still incompletely defined.

Amino Acid Sequence↗

PubMed1994

Expression strategy of the M genome segment of Hantaan virus.

The medium (M) genome segment of Hantaan virus encodes the envelope glycoproteins, G1 and G2, in a continuous open reading frame with a gene order of 5'-G1-G2-3' with respect to the virus-complementary sense RNA. Because potential translation initiation codons and amino acids constituting typical signal sequences precede both the G1 and G2 genes, we sought to determine if G1 and G2 can be expressed independently. To investigate translational requirements for G1 and G2, we constructed M segment genes in which portions of the coding information were mutated or deleted, and transiently expressed these genes in eukaryotic cells by using a vaccinia virus/T7 RNA polymerase system. We found that G2 expression can occur by ribosomal access to the translation initiation codon preceding the G2 signal sequence (nucleotides 1934-1936), but that other upstream AUG codons cannot be used as efficiently. The presence of this codon, however, was not required for G2 expression because changing nucleotides 1934-1936 to CUG, GCG or AUG did not abrogate expression of G2. We also found that leaky ribosomal scanning, rather than internal initiation of translation was the most likely explanation for the observed independent translational initiation of G2, but that not all upstream, in-frame AUGs could serve as initiator codons. To assess the requirement for a continuous open reading frame for G1 and G2 expression, we expressed a gene which had G1 and G2 coding information in different reading frames. Although G1 was expressed at apparently normal levels, little or no G2 was expressed. In contrast, only G2 was expressed from a gene in which the carboxy-terminal G1 coding information was deleted and the remaining, truncated G1 was placed out of frame with respect to G2. These data suggest that reinitiation of translation may occur under some, but not all, circumstances when the polyprotein coding information is perturbed. Our results are consistent with biogenesis of G1 and G2 primarily or entirely according to the ribosomal scanning model.

Animals↗

PubMed2004

African horsesickness virus serotyping and identification of multiple co-infecting serotypes with a single genome segment 2 RT-PCR amplification and reverse line blot hybridization.

Since protection against African horsesickness (AHS) is serotype-specific, rapid serotyping of AHSV is crucial to identify the correct vaccine serotype for efficient control of the spread of AHS outbreaks, especially when they occur in non-endemic regions. This paper describes the first one-day serotyping procedure that requires only a single RT-PCR and hybridization and which can identify multiple serotypes in mixed infections in one assay. The same region of genome segment 2 of all nine AHSV serotypes is amplified in a single RT-PCR. A universal primer set, designed to amplify the 5'-terminal 521-553bp of genome segment 2 of all of the nine AHSV serotypes with one reaction, was used to generate serotype-specific probes from dsRNA prepared from infected tissue cultures or organ samples. These probes hybridized serotype-specifically with immobilized genome segment 2 cDNA of the nine AHSV reference serotypes in a checkerboard reverse line blot format. All nine AHSV reference and the seven vaccine strains and field viruses isolated up to 28 years apart could be serotyped accurately within a day. The sensitivity of the method is 1pg dsRNA which is sufficient to serotype AHSV directly from lung and spleen specimens of infected horses.

African Horse Sickness↗

PubMed1988

The sequence of the reovirus serotype 3 L3 genome segment which encodes the major core protein lambda 1.

We present the sequence of reovirus serotype 3 (strain Dearing) genome segment L3 which encodes protein lambda 1, one of the two major components of the core shell. The genome segment is 3896 nucleotides long, with 5'- and 3'-noncoding regions of 13 and 181 nucleotides, respectively. Protein lambda 1 is 1233 amino acids long. It is a slightly acidic protein, with a predicted alpha-helix and beta-sheet content of 23.6 and 28.3%, respectively. Its rather low predicted alpha-helix contact is consistent with its being a structural protein. The 123 amino acid long region at its amino terminus is very hydrophilic and contains three alpha-helical regions, one being 26 amino acids long. Protein lambda 1 contains two functional motifs. The first is a nucleotide binding site -TKGKSSG- starting at residue 8, the other is a "zinc finger" motif centered around amino acid residue 194. This suggests that protein lambda 1 functions during the transcription of either dsRNA into plus strands or of plus strands into minus strands, or during both. It displays no significant sequence similarity to any protein sequence in the GenBank data base.

Amino Acid Sequence↗