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PubMed1999

Packaging and replication regulation revealed by chimeric genome segments of double-stranded RNA bacteriophage phi6.

Bacteriophage phi6 has a double-stranded RNA genome composed of three linear segments, L, M, and S. The innermost particle in the virion of phi6, like in the other dsRNA viruses, is an RNA-dependent RNA polymerase complex, which carries out all the functions needed for the replication of the viral genome. Empty polymerase complexes can package the single-stranded copies of the viral genome segments, replicate the packaged segments into double-stranded form (minus strand synthesis), and then produce new plus strands (transcripts) from the double-stranded RNA templates. The three viral genomic segments contain unique packaging signals at their 5' ends, and minus strand synthesis initiation is dependent on the sequence at the 3' end. Here we have constructed chimeric segments that have the packaging signal from one segment and the minus strand synthesis initiation signal from another segment. Using purified recombinant polymerase complexes and single-stranded/chimeric and original RNA segments, we have analyzed the packaging and replication regulation operating in in vitro conditions. We show that the 5' end of the L genome segment in single-stranded form is needed to switch from the packaging to the minus strand synthesis and the same sequence is required in double-stranded form to switch on plus strand synthesis. In addition we have constructed deletions to the M segment to analyze the possible regulatory role of the internal noncoding area of this segment.

Bacteriophage phi 6↗

PubMed1992

In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA.

The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.

Capsid↗

PubMed1991

Evidence of genome segment 5 reassortment in bluetongue virus field isolates.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.

Animals↗

PubMed1979

5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus.

EXtensive nucleotide sequence analysis of the 5' and the 3' terminal of the RNA segments of the genome of fowl plague virus, an avian strain of influenza virus, confirms the presence of a common sequence at the 5' terminus of each segment and a common sequence at the 3' terminus of each segment. Between the ends of each individual segment there is a complementary sequence which may be important in the control of transcription and replication of the genome. In addition, the probable sites of initiation of translation of fowl plague virus mRNA are indicated along with the corresponding NH2-terminal amino acid sequences of the virus polypeptides.

Base Sequence↗

PubMed2003

Assembly into single-shelled virus-like particles by major capsid protein VP1 encoded by genome segment S1 of Bombyx mori cypovirus 1.

The major capsid protein VP1 encoded by genome segment S1 of Bombyx mori cypovirus 1 was expressed in a baculovirus system. In the absence of any other capsid proteins, VP1 was found to assemble into single-shelled virus-like particles. The VP1 particles were more sensitive to acidic conditions than were intact particles.

Baculoviridae↗

PubMed2003

Molecular analysis of Fiji disease virus genome segments 5, 6, 8 and 10.

The complete sequences of Fiji disease virus (FDV) genome segments 5 (S5), S6, S8 and S10 were obtained and comprised 3150 nt, 2831 nt, 1959 nt and 1819 nt, respectively. Each segment contained a single ORF which encoded putative proteins of 115 kDa, 97 kDa, 69 kDa and 63.0 kDa, respectively. The putative amino acid sequences encoded by S5 and S6 contained putative leucine zipper motifs while FDV S5 and S8 each contained an ATP-GTP-binding motif. At the amino acid level, FDV S5, S6, S8 and S10 showed most similarity to the corresponding segments of Rice black-streaked dwarf virus. Based on sequence similarities, it is predicted that FDV S8 encodes a minor core protein, while FDV S10 encodes an outer capsid protein. The evolutionary relationships of FDV to other reoviruses are discussed.

Capsid Proteins↗

PubMed2003

Identification of parental origin of cognate dsRNA genome segment(s) of rotavirus reassortants by constant denaturant gel electrophoresis.

Rotaviruses are the single most important etiologic agents of severe diarrhea in infants and young children worldwide. They possess a triple capsid morphology and a genome of 11 segments of double-stranded (ds) RNA. During the course of the development of various live, attenuated reassortant rotavirus vaccines, we often experienced difficulty in identifying the parental origin of certain genome segment(s) of a reassortant vaccine candidate. Various assays have been utilized for determination of the parental origin of reassortant virus genes, including polyacrylamide gel electrophoresis (PAGE), DNA and/or RNA hybridization assays and gene sequence analysis. The traditional PAGE is simple and easy to perform, however, it is common to find that certain cognate dsRNA segment(s) cannot be differentiated by this assay due to a high degree of sequence homology among different rotavirus strains. Constant denaturant gel electrophoresis (CDGE) is one of several methods that have been used to screen DNA fragments for small sequence changes or point mutations. By using the CDGE, we were successful in partially denaturing rotavirus dsRNA thereby changing the physical properties of the genome segment(s) in the gel and thus differentiating the cognate genome segment(s) of rotavirus reassortants. The CDGE provides a simple and reliable assay system for identification of parental gene origins of a rotavirus reassortant.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

Nucleotide sequences of genome segments S6, S7 and S10 of Dendrolimus punctatus cypovirus 1.

The nucleotide sequences of genome segments S6, S7 and S10 of Dendrolimus punctatus cypovirus 1 Hunan I (DpCPV-HN(I)) and DpCPV-HN(I)-Se(3) (DpCPV-HN(I) passed three times in Spodoptera exigua) were determined. Segment S10 was 944 nucleotides in length and encoded a polyhedrin of 248 amino acids (28,439 Da). Only two nucleotide mutations were found between DpCPV-HN(I) S10 and DpCPV-HN(I)-Se3 S10, and the deduced amino acid sequences of the polyhedrin proteins were identical. Segment S7, 1 501 nucleotides, encoded a protein of 448 amino acids ( approximately 50 kDa; p50). Thirty-one nucleotide mutations were found between DpCPV-HN(I) S7 and DpCPV-HN(I)-Se3 S7, but these resulted in only four amino acid changes. DpCPV-HN(I) S6 encoded a protein of 561 amino acids (63,688 Da; p64). The amino acid sequence of p64, had a high leucine content (10%), and contained a leucine zipper motif and one ATP/GTP-binding site motif.

Animals↗

PubMed1994

Nucleotide sequence of Nilaparvata lugens reovirus genome segment S8 coding for the major outer capsid protein.

The complete nucleotide sequence of genome segment 8 (S8) of Nilaparvata lugens reovirus (NLRV) was determined. It consisted of 1802 nucleotides containing a long open reading frame (562 amino acids), which was expressed in Escherichia coli as a fusion protein. The expressed S8 product, a 62K protein, was detected by Western blotting using IgG directed against intact NLRV particles. This result indicates that S8 encodes the major outer capsid protein of NLRV. The protein exhibited 18.6% amino acid sequence identity with the predicted translation product of S10 of rice black-streaked dwarf virus.

Amino Acid Sequence↗

PubMed1996

The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67,348 polypeptide encoded by genome segment 8.

The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined. RRSVS8 is 1914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1810 which is capable of encoding a protein of M(r) 67,348. The N-terminal amino acid sequence of a approximately 43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence. These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage. Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K. Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles. Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K. Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product. Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products. These data indicate that S8 encodes a structural polypeptide, the majority of which is auto-catalytically cleaved to 26K and 46K proteins. The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a approximately 43K major capsid protein.

Amino Acid Sequence↗

PubMed2001

Genome segment S8 of grass carp hemorrhage virus encodes a virion protein.

The complete nucleotide sequence of the genome segment S8 of grass carp hemorrhage virus (GCHV) was determined from cDNA corresponding to the viral genomic RNA. It is 1,287 nucleotides in length and contains a large open reading frame that could encode a protein of 409 amino acids with a predicted molecular mass of 44 kD. The S8 was expressed using the pET fusion protein vector and detected by Western blotting analysis using the chicken egg IgY against intact GCHV particles, indicating that S8 encodes a virion protein. Amino acid sequence comparisons revealed that the protein encoded by S8 is closely related to protein sigma2 of mammalian reovirus, suggesting that the deduced protein of S8 is an inner capsid protein.

Amino Acid Sequence↗

PubMed1990

cDNA cloning of each genomic segment of the group B rotavirus ADRV: molecular characterization of the 11th RNA segment.

The group B noncultivatable rotavirus, ADRV, was purified from infected stool specimens. Double-stranded RNA was extracted, polyadenylated, reverse transcribed into cDNA, and cloned into plasmid vector pAT153. Each cDNA clone hybridized to a single ADRV RNA segment and cDNA clones of each genomic RNA segment were identified. Sequencing of genomic RNA and a full-length cDNA clone (AD63) determined that ADRV RNA segment 11 is 631 bases in length and contains a single open reading frame of 170 amino acids with a calculated molecular weight of 19.9 kDa and a pl of 6.2. The RNA 5'- and 3'-termini contain untranslated regions of 58 and 63 bases, respectively, and are complementary to each other. A comparison of encoded ADRV gene 11 amino acids with the NS26 protein of group A rotaviruses demonstrates a distant similarity between the two proteins. Further analysis and use of these ADRV clones should aid in determining the gene coding assignments for group B proteins as well as for diagnostic detection of group B or ADRV-specific nucleic acids in specimens.

Amino Acid Sequence↗

PubMed2000

Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae).

The genome segments 1, 2, and 3 of the grass carp reovirus (GCRV), a tentative species assigned to genus Aquareovirus, family Reoviridae, were sequenced. The respective segments 1, 2, and 3 were 3949, 3877, and 3702 nucleotides long. Conserved motifs 5' (GUUAUUU) and 3' (UUCAUC) were found at the ends of each segment. Each segment contains a single ORF and the negative strand does not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VP1 might represent the viral guanylyl/methyl transferase (involved in the capping process of RNA transcripts) and VP3 the NTPase/helicase (involved in the transcription and capping of viral RNAs). The highest amino acid identities (26-41%) were found with orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses, and orthoreoviruses. It should also permit to precise the taxonomic status of these different viruses.

Animals↗

PubMed1994

Comparison of nucleotide sequences of M genome segments among Seoul virus strains isolated from eastern Asia.

The nucleotide sequences of the M genome segments of three Seoul virus strains (KI strains) which were isolated from urban rats inhabiting the same enzootic focus between 1983 and 1988 were compared. The viral cDNAs were amplified by PCR and were directly sequenced. The nucleotide sequences of KI strains were extremely homologous regardless of isolation year (less than 10 substitutions in 3651 nucleotides, less than 4 substitutions in 1133 amino acids). In addition, the nucleotide sequence of the KI strain isolated in 1983 (KI-83-262) was also quite similar to that of other Seoul viruses, which were isolated from laboratory rats in Japan (strain SR-11, 98.1% and B-1 strain, 96.5%), from an urban rat in Korea (Seoul 80-39, 96.5%) and from an urban rat in China (R22 strain, 93.4%). All possible N-glycosylation sites in the deduced amino acid sequences were conserved among all Seoul viruses examined. The nucleotide and amino acid sequences of Seoul virus strains were highly conserved although they were isolated from various districts of eastern Asia. These results indicate the genetic stability of Seoul virus strains maintained under a natural environment and the homology of Seoul viruses isolated from various districts of eastern Asia. The relationship among Seoul virus strains isolated from eastern Asia was compared by phylogenetic analysis.

Amino Acid Sequence↗

PubMed2002

Phylogeny of New World arenaviruses based on the complete coding sequences of the small genomic segment identified an evolutionary lineage produced by intrasegmental recombination.

Previous studies suggested that the small genomic segments (S-RNA) of the South American arenaviruses (SA-AVs) represent three phylogenetic lineages (designated A, B, and C) and indicated that the S-RNA of Whitewater Arroyo virus (WWAV) (a North American arenavirus [NA-AV]) is a product of genetic recombination between a lineage A and lineage B virus. The purpose of this study was to extend our knowledge on the phylogenetic relationships between WWAV, the two other NA-AVs (Tamiami and bear canyon), and the 15 SA-AVs. Therefore, we determined the complete sequence of the S-RNA of nine arenaviruses previously uncharacterized or sequenced only partially. Phylogenetic analyses of the two complete coding regions indicated that the S-RNA of the three NA-AVs have descended from a single ancestral virus, which was the product of recombination between a lineage A and lineage B arenavirus. No such evidence for genetic recombination was found in cupixi virus (a novel arenavirus isolated from a wild rodent captured in Northeastern Brazil) or the 14 other SA-AVs. The recombinant nature of the S-RNA of NA-AVs distinguishes them from the SA-AVs, and thus, indicates that the NA-AVs represent a fourth phylogenetic lineage in the Tacaribe serocomplex.

Amino Acid Sequence↗

PubMed1988

Sequence analysis and in vitro expression of a cDNA clone of genome segment 5 from bluetongue virus, serotype 1 from South Africa.

A full length copy of genome segment 5 of bluetongue virus serotype 1 from South Africa (BTV-1SA) was assembled from two incomplete cDNA clones. The complete nucleotide sequence was determined (1635 nucleotides in length) and an open reading frame coding for 527 amino acids was found, which was flanked by a 5' non-coding region of 25 nucleotides and a 3' non-coding region of 29 nucleotides. The cDNA clone was transferred to an Sp6 expression vector from which an RNA transcript was obtained. This transcript, when translated in vitro in a reticulocyte lysate system, produced a protein that co-migrated during electrophoresis with both protein VP5 from disrupted virus particles and VP5 translated from denatured viral dsRNA. The protein synthesized from the cDNA clone was precipitable with antisera raised against BTV-1SA virus particles and with antisera raised against a synthetic peptide, the sequence of which was obtained from the predicted amino acid sequence of BTV-1SA protein VP5. These antisera also precipitated protein VP5 translated from denatured viral dsRNA. Collectively these data indicate that the cDNA clone encodes an authentic VP5 protein product. The amino acid sequence of BTV-1SA VP5, when compared to other published sequences for VP5, contained highly conserved regions interrupted by variable domains. If two isolates of the same serotype are compared, (BTV-1SA and BTV-1AUS) only two variable regions are apparent. However, if the amino acid sequences of VP5 from two different serotypes are compared, (BTV-1SA and BTV-10), eight variable regions are detectable (two of which are in the same position as the variable regions within a serotype). The implications of these variations in the outer coat protein, VP5, are discussed.

Amino Acid Sequence↗

PubMed1987

A comparison of six different bluetongue virus isolates by cross-hybridization of the dsRNA genome segments.

The relationship between six different isolates of BTV was analyzed by cross-hybridization of genomic dsRNA using blotting and probe techniques (using an alkali fragmented probe made from BTV dsRNA). The viruses compared in this way included BTV serotype 1 from South Africa, serotypes 3 and 4 from Cyprus, serotype 10 from North America, and serotypes 1 and 20 from Australia. Under the hybridization and washing conditions used, which were calculated to allow stable duplex formation between RNA molecules containing greater than 90% sequence homology, two of the genome segments (segments 2 and either 5 or 6, which encode the two major outer capsid proteins VP2 and VP5) appeared to contain serotype-specific RNA sequences. Significant cross-hybridization between these segments from different serotypes was detected only with serotypes 4 and 20, which are known to have a particularly close antigenic relationship. The amounts of homologous sequence that were detected in segments other than 2 and 5 between different viruses indicated some correlation between their geographical origins and a degree of relatedness, which is independent of the virus serotype. High levels of sequence homology were detected between the isolates from Cyprus and Africa and to a slightly lesser extent from North America, suggesting a common ancestry. These results also indicated that within the limited number of viruses studied, the Australian isolates form a separate interrelated group of bluetongue viruses.

Bluetongue virus↗

PubMed1993

Magnetic bead capture of expressed sequences encoded within large genomic segments.

Magnetic bead capture utilizes biotin-streptavidin magnetic bead technology to isolate cDNAs rapidly from large genomic intervals, giving several thousand-fold enrichment of the selected cDNAs. The technique can allow parallel analysis of several large genomic segments of varying complexities and can be applied to the isolation of expressed sequences from various tissue sources.

Bacterial Proteins↗