Some highlights of virus research in 1990.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R M Elliott
Publications and source records attributed to R M Elliott.
Expression of functional Bunyamwera virus L protein by recombinant vaccinia viruses.
A cDNA containing the complete coding sequence of the Bunyamwera virus (family Bunyaviridae) L genome segment has been constructed and cloned into two recombinant vaccinia virus expression systems. In the first, the L gene is under control of vaccinia virus P7.5 promoter; in the second, the L gene is under control of the bacteriophage T7 phi 10 promoter, and expression of the L gene requires coinfection with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. Both systems express a protein which is the same size as the Bunyamwera virus L protein and is recognized by a monospecific L antiserum. The expressed L protein was shown to be functional in synthesizing Bunyamwera virus RNA in a nucleocapsid transfection assay: recombinant vaccinia virus-infected cells were transfected with purified Bunyamwera virus nucleocapsids, and subsequently, total cellular RNA was analyzed by Northern (RNA) blotting. No Bunyamwera virus RNA was detected in control transfections, but in cells which had previously been infected with recombinant vaccinia viruses expressing the L protein, both positive- and negative-sense Bunyamwera virus S segment RNA was detected. The suitability of this system to delineate functional domains within the Bunyamwera virus L protein is discussed.
Animals↗
Molecular biology of the Bunyaviridae.
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Amino Acid Sequence↗
Some highlights of virus research in 1989.
Explore the source record for details and available documents.
Amino Acid Sequence↗
A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones.
An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described. Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria. After confluent lysis, recombinant bacteriophage was extracted with SM buffer. Bacterial debris was removed by centrifugation. A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts. The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5. Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform. Such lambda gt11 DNA was readily digested with EcoRI. Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library. We have used this procedure to isolate a full-length alkaline phosphatase cDNA. The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.
Bacteriophage lambda↗
Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae.
The complete nucleotide sequence of the large (L) genome segment of Bunyamwera virus has been determined from overlapping cDNA clones. The segment is 6875 nucleotides long and has a base composition of 29.8% A, 17.9% C, 15.4% G, and 36.9% U. Eighteen of the terminal 19 nucleotides at the 3' and 5' ends are complementary. In the viral-complementary (+ sense) RNA there is a single long open reading frame (ORF) from AUG at bases 51-53 to a UAG stop codon at bases 6765-6767; this ORF encodes a polypeptide of 2238 amino acids (MW 259,000), corresponding to the L protein which has been mapped to the L RNA segment by analysis of reassortants of Bunyamwera, Batai, and Maguari viruses. The amino-terminal 46 amino acids of the L protein show strong homology (63% identity) with the amino-termini of ORFs predicted from limited sequence analysis of the L segments of La Crosse and snowshoe hare bunyaviruses. Comparison with the polymerase proteins encoded by other negative-strand viruses showed weak homology with part of the influenza virus PB1 protein, but no homology was detected with the other influenza virus polymerase proteins nor with the L proteins of arenaviruses, paramyxoviruses, and rhabdoviruses. At the 5' end of genomic (- sense) RNA there is an AUG-initiated ORF potentially encoding a protein of 14,700; the significance of this ORF is unknown at present.
Amino Acid Sequence↗
Nucleotide sequence and expression of the small (S) RNA segment of Maguari bunyavirus.
The small (S) RNA segment of the Maguari bunyavirus genome has been cloned as cDNA and its nucleotide sequence determined. The nucleocapsid protein, N, (Mr 26K) and a nonstructural protein, NSs, (Mr 11K), are encoded in overlapping reading frames, similar to other bunyavirus S RNA segments. In addition, a third AUG-initiated open reading frame encoding a 9.3K protein was observed. All three polypeptides were translated in cell free systems programmed with RNA transcribed in vitro from the cDNA subcloned downstream of a bacteriophage T7 promoter. The effects on expression of subcloning parts of the cDNA and by site-specific mutagenesis are discussed in relation to the scanning model of initiation of translation. A recombinant baculovirus has been constructed to express the Maguari virus S segment gene products. The N protein was efficiently expressed in infected cells, and a significant amount was in a soluble form. We could not detect the synthesis of NSs nor the 9.3K protein, and the reasons for this are discussed. The 9.3K protein has not been found in Maguari virus-infected cells and so the question of its functional significance remains open.
Amino Acid Sequence↗
Nucleotide sequence analysis of the small (S) RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae.
The nucleotide sequence of the small (S) RNA segment of the Bunyamwera virus genome has been determined. The S RNA is 961 bases in length and, in common with other bunyaviruses, encodes two proteins, N and NSs, in overlapping reading frames. A six-way alignment of the amino acid sequences of the N and NSs proteins of viruses representing three serogroups within the Bunyavirus genus indicates regions which are strongly conserved, and provides targets for future analysis of protein function.
Amino Acid Sequence↗
Persistent infection of Aedes albopictus C6/36 cells by Bunyamwera virus.
Two cell lines persistently infected with Bunyamwera virus have been established from the C6/36 clone of Aedes albopictus cells. The cells express Bunyamwera virus antigens as detected by immunofluorescence and are resistant to superinfection with Bunyamwera virus and other bunyaviruses, but not Dugbe virus (Nairovirus) nor vesicular stomatitis virus. The virus released from the persistently infected cells developed an altered cloudy or "bull's-eye" plaque morphology with increasing passage level, and a greater temperature sensitivity at 39.5 degrees than standard virus. The persistent virus interfered strongly with the replication of standard Bunyamwera virus in normal C6/36 cells and to a much lesser extent in BHK cells. Interference was not noted with other bunyaviruses or vesicular stomatitis virus. The persistent virus from one cell line, C6/36-PI LO, had a slower migrating nucleocapsid protein on polyacrylamide gels. Analysis of the RNA in persistently infected cells or in persistent virus by Northern blot hybridization with cloned cDNA probes showed that the major viral RNA species was the S segment, while the L and M RNA segments were barely detectable. Our results indicate that Bunyamwera virus can readily establish persistent infections in mosquito cells, and that persistence is accompanied by the generation of viruses with variable genetic and phenotypic characteristics.
Aedes↗
Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of structural features in the Bunyavirus glycoprotein gene product.
The complete nucleotide sequence of the Bunyamwera virus M RNA segment was determined from four overlapping cDNA clones and by primer extension. The RNA segment is 4458 bases in length, and encodes a single gene product in the viral complementary RNA. The predicted protein is 1433 amino acids long (mol wt 162,065), contains four potential glycosylation sites, and is relatively cysteine rich. It is presumed that the three proteins G1, G2, and NSM which have been mapped to the M RNA segment are synthesized as a precursor polyprotein which is subsequently proteolytically cleaved. A putative hydrophobic signal sequence at the amino terminus and a hydrophobic anchor sequence at the carboxy terminus of the predicted protein have been identified, in addition to internal regions of hydrophobicity of unknown function. The nucleotide and amino acid sequences of the Bunyamwera virus M segment have been compared with those of the snowshoe hare virus M segment (Y. Eshita and D. H. L. Bishop, Virology 137, 227-240, 1984). Common features include the overall architecture of the RNAs, single cysteine-rich primary gene products, and conservation of hydrophobic domains in the gene products. When aligned the amino acid sequences are 43% homologous, and 66 of 70 cysteine residues can be matched. The evolutionary significance of these findings is discussed.
Amino Acid Sequence↗
Characterization of the mutations responsible for the electrophoretic mobility differences in the NS proteins of vesicular stomatitis virus New Jersey complementation group E mutants.
Temperature-sensitive (ts) mutants of vesicular stomatitis virus, New Jersey serotype, classified in complementation group E contain lesions in the NS gene, which manifest as marked electrophoretic mobility differences of the mutant NS proteins in SDS-polyacrylamide gels. We have cloned full-length cDNA copies of the mutant NS mRNAs, and have determined their nucleotide sequences. tsE1 and tsE3 had single nucleotide changes, and tsE2 had two nucleotide changes, compared to the wild-type NS gene. Three of the mutations were clustered in a region of 18 nucleotides. All the nucleotide differences resulted in amino acid substitutions, which in each case changed the charge of the amino acid concerned. Analysis of the wild-type and mutant NS protein sequences by the method of Chou & Fasman indicated that single amino acid substitutions can radically alter the predicted secondary structure, and these data are discussed in relation to the observed electrophoretic mobility differences.
Amino Acid Sequence↗
Conservation of potential phosphorylation sites in the NS proteins of the New Jersey and Indiana serotypes of vesicular stomatitis virus.
A full length cDNA copy of the NS mRNA of the Missouri strain (Hazelhurst subtype, New Jersey serotype) of vesicular stomatitis virus (VSV) has been cloned and sequenced. The mRNA is 856 nucleotides long (excluding polyadenylic acid) and encodes a protein of 274 amino acids (mol. wt. 31 000). Comparison with the NS gene of the Ogden strain (Concan subtype, New Jersey serotype) showed 15% difference at the nucleotide level and 10% difference at the amino acid level; the majority of the changes were located in the 3' half of the mRNA. Comparison with the NS genes of two strains representing the Indiana serotype showed about 50% nucleotide and 33% amino acid sequence homology between the serotypes. In a four-way comparison of the proteins, two regions of higher homology were noted which may be of functional importance. Eighteen potential phosphorylation sites (Ser or Thr) were conserved between the four proteins; five of these sites correspond to the residues which have been suggested to be constitutively phosphorylated and may be essential for NS activity.
Base Sequence↗
A survey of respiratory syncytial virus and parainfluenza virus type 3 neutralising and immunoprecipitating antibodies in relation to Paget disease.
The aetiology of Paget disease of bone has not been established but certain features have suggested involvement of a parainfluenzalike virus. To seek further evidence of the possible role of paramyxoviruses in Paget disease we have surveyed the presence of neutralising and immunoprecipitating antibodies to both respiratory syncytial virus and parainfluenza virus type 3 in the sera of patients attending a bone disease clinic. These two viruses were implicated by the sporadic observation of viral antigen in individual nuclei of osteoclasts in Paget disease bone lesions. A total of 315 samples were obtained from 177 patients attending the clinic during 1 year. Thirty-six of the patients had confirmed Paget disease and the remainder other conditions. All sera possessed neutralising activity to both viruses. The mean titres for each virus were similar in patients with Paget disease and those with other conditions whether matched or not. In the case of respiratory syncytial virus the neutralising titres were distributed closer to the mean in the Paget group and showed little variation in repeat samples taken over periods of up to 1 year in contrast to the greater variability of the control group. The antigenic specificity of 20 age- and sex-matched sera from each group was examined by immunoprecipitation. No significant differences were observed between Paget and non-Paget patients. These results do not provide confirmation of involvement of either virus in Paget disease, but the serological data suggest that persistent infection with respiratory syncytial virus can occur.
Adolescent↗
Identification of nonstructural proteins encoded by viruses of the Bunyamwera serogroup (family Bunyaviridae).
The proteins synthesized in BHK cells infected with nine members of the Bunyamwera serogroup (family Bunyaviridae, Bunyavirus genus) were analyzed by polyacrylamide gel electrophoresis. In addition to the virus structural proteins, a number of virus-coded nonstructural proteins were detected. One protein, designated NS1, was shown to be related to the nucleocapsid protein by one-dimensional peptide mapping. A second protein, NS2, was mapped to the M RNA segment by gel electrophoretic analysis of the proteins synthesized in cells infected with reassortants of Batai, Bunyamwera, and Maguari viruses of known genotype. A third protein, NS3, was mapped to the S RNA segment by its pattern of labeling with [35S]cysteine in cells infected with reassortant viruses: the NS3 protein was only labeled when the S RNA segment of Bunyamwera virus was present. The mapping of NS3 was confirmed by in vitro translation of mRNAs which hybridized to recombinant plasmids containing S gene-specific sequences.
Animals↗
Protein and nucleic acid analyses of influenza C viruses isolated from pigs and man.
The virus-coded proteins and the genomes of influenza C virus isolates obtained from Chinese pigs in 1981-1982 and of human influenza C virus strains isolated between 1947 and 1981 were compared. Using SDS polyacrylamide gel electrophoresis and one-dimensional peptide mapping we found the virus-coded proteins of the pig influenza C viruses to be similar to those of human influenza C virus strains. The sizes of the genomes of human and pig influenza C viruses were indistinguishable. Genome analysis by oligonucleotide (ON) mapping revealed that the genomes of the pig influenza C viruses were very similar to but not identical with those of human influenza C virus strains. ON changes were found scattered over the whole genome. ON mapping of isolated segments of several influenza C virus strains suggested that two pig strains (C/P/B/10/81 and C/P/B/32/81) are related by a reassortment event which is likely to have occurred in nature. The rate of genome variation in influenza C viruses seemed to be similar to that seen in influenza B, and slower than that recorded for influenza A viruses.
Animals↗
The proteins and RNAs specified by Clo Mor virus, a Scottish Nairovirus.
The proteins and RNAs of Clo Mor virus have been analysed. Virus-specific proteins in infected cells had previously been identified by isotopic labelling and radioimmunoprecipitation; these were three glycoproteins (mol. wt. 115K, 90K and 80K) and an unglycosylated nucleocapsid (N) protein (50K). We have performed pulse-chase experiments which indicated that the 115K protein is processed to give the 90K and 80K proteins, while a 45K protein was detected in released virions after prolonged chase. Translation in vitro of mRNA extracted from Clo Mor virus-infected cells resolved only the N protein. Three species of RNA were extracted from Clo Mor virus intracellular nucleocapsids and have been designated L (11000 to 13000 bases), M (6300 bases) and S (1900 bases). The processing of viral proteins and the sizes of RNAs are characteristic of the Nairovirus genus of the family Bunyaviridae.
Bunyaviridae↗
Synthesis of bunyavirus-specific proteins in a continuous cell line (XTC-2) derived from Xenopus laevis.
The XTC-2 cell line, derived from Xenopus laevis, supported the replication of representative viruses from each of the four genera in the family Bunyaviridae. Generally, viral titres were higher in XTC-2 cells than in other susceptible cell lines, and for some viruses plaques were detected earlier in XTC-2 cells. The XTC-2 cell line permitted comparative analyses of bunyavirus-specific protein synthesis. The patterns of synthesis of viral proteins, characteristic of each of the genera, were observed with representative viruses. These studies provided biochemical characterization of two Scottish isolates, which support the inclusion of Clo Mor virus in the Nairovirus genus and St Abb's Head (M349) virus in the Uukuvirus genus.
Animals↗
The proteins and RNAs of St. Abb's Head virus, a Scottish Uukuvirus.
The proteins and RNAs of St. Abb's Head virus have been analysed, and were found to be characteristic of the Uukuvirus genus of the family Bunyaviridae. Two glycoproteins, G1 and G2 (mol. wt. 62K and 75K), and a nucleocapsid protein, N (mol. wt. 25K), were detected in infected cells by immunoprecipitation; the synthesis of N preceded the synthesis of G1 and G2. The glycoproteins were relatively cysteine-rich compared to the N protein, and the unglycosylated forms of G1 and G2 (using the inhibitor tunicamycin) had a mol. wt. of about 58K. Translation in vitro of mRNA from infected cells gave two immunoprecipitable products which are thought to be equivalent to N and G1/G2. Three RNA species were found in St. Abb's Head virus nucleocapsids, and were estimated to be 8500 bases (L), 3600 bases (M) and 1900 bases (S) in length. At least one additional virus-specific RNA species was detected in infected cells. The similarity between the proteins and RNAs of St. Abb's Head virus and Uukuniemi virus (the prototype of the genus) is discussed.
Animals↗