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D V Sangar

Publications and source records attributed to D V Sangar.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1984

Multiple proteases in foot-and-mouth disease virus replication.

Translation of foot-and-mouth disease virus RNA in a rabbit reticulocyte lysate for short time intervals resulted in the production of the peptides P20a , P16, and P88 (Lab, Lb, and P1) (R. R. Rueckert , Recommendations of the 3rd European Study Group on Molecular Biology of Picornavirus, Urbino , Italy, 1983). If further translation was prevented, the structural protein precursor P88 was not cleaved, even after prolonged incubation. This result indicates that the mechanism of the cleavage between P20a -P16 and P88 and of that between P88 and P52 (P2) differs from the mechanism of the secondary cleavages which produce the structural proteins. Furthermore, treatment of foot-and-mouth disease virus-infected cells with the protease inhibitor D-valyl phenylalanyl lysyl chloromethyl ketone prevented the in vivo cleavage between P20a -P16 and P88 but had no effect on any of the other cleavage events. These results suggest that the cleavage of the foot-and-mouth disease virus polyprotein utilizes two different host proteases.

Animals↗
PubMed1981

Removal of the genome-linked protein of foot-and-mouth disease virus by rabbit reticulocyte lysate.

Rabbit reticulocyte lysate cleaves the genome-linked protein VPg from foot-and-mouth disease virus (FMDV) RNA. This activity could be reliably monitored since removal of the protein resulted in a change in migration in polyacrylamide gels of the small specific 5' and fragment of the RNA (S fragment). The unlinking activity cleaved the bond between the tyrosine residue of VPg and the RNA to leave a 5' phosphate on the RNA. The 5' sequence of the RNA from which VPg had been removed by rabbit reticulocyte lysate was the same as that of FMDV mRNA isolated from infected cells. VPg released from the RNA was rapidly degraded by the rabbit reticulocyte lysate to material which eluted with the inclusion volume of a Sepharose 6B column and partitioned to the aqueous phase during phenol extraction. The unlinking activity was inhibited by heating the lysate to 56 degrees C, by sodium dodecyl sulfate (SDS), EDTA, and Zn2+ ions but was unaffected by reducing agents, a translation inhibitor, and a number of protease and RNase inhibitors.

Animals↗
PubMed1980

Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA.

An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.

Aphthovirus↗
PubMed1980

Heterogeneity of the genome-linked protein of foot-and-mouth disease virus.

The genome-linked protein of foot-and-mouth disease virus was examined by electrofocusing in polyacrylamide gels. Two proteins of different charge and amino acid composition were found. The tryptic peptide maps of the proteins were dissimilar. The possible relationship between the two proteins is discussed.

Aphthovirus↗
PubMed1978

A re-appraisal of the biochemical map of foot-and-mouth disease virus RNA.

The proteins induced by infection of BHK 21 cells with foot-and-mouth disease virus have been compared by tryptic peptide analysis. The results indicate that there are three primary products 5'--P88, P52, P100--3'. The polypeptide P56, which we considered previously to be a primary product, is derived from the region of the genome that codes for P100. The results indicate that there are alternative cleavage pathways of P100, the polypeptide coded for by the 3' end of the genome.

Aphthovirus↗
PubMed1977

Immunogenic and cell attachment sites of FMDV: further evidence for their location in a single capsid polypeptide.

Chymotrypsin cleaves only one of the four major polypeptides of foot-and-mouth disease virus (FMDV serotype O) in situ. This polypeptide (VP1, mol. wt. 29 X 10(3) was first cleaved into fragments of mol. wt. 20 and 9 X 10(3) and further cleavage could be prevented by the addition of a large excess of bovine serum albumin. The infectivity of the virus particles at this stage was the same as that of the intact virus although the rate of attachment to BHK 21 cells was slower and the immunogenic activity was reduced. If hydrolysis was allowed to continue, VP1 was cleaved into fragments with mol. wt. 18 and less than 9 X 10(3), similar to those obtained with trypsin and the virus particles then had a greatly reduced infectivity and a lower immunogenicity. Treatment of strains from five other serotypes of the virus with the two enzymes cleaved only VP1 in each instance and there was a corresponding loss of infectivity. The results are discussed in relation to the location and biological activity of the virus polypeptides.

Animals↗
PubMed1977

Biochemical mapping of the foot-and-mouth disease virus genome.

Four primary cleavage products, mol. wt. 10(3) X 100, 88, 56 and 52 (P100, P85, P56 and P52 respectively) are present in BHK 2I cells infected with foot-and-mouth disease virus (FMDV). However, no precursor polyprotein equal to the sum of their mol. wt. was detected, even when amino acid analogues and proteolytic enzyme inhibitors were used. Three of the primary products were shown to cleave to smaller polypeptides, including the capsid polypeptides of the virus. Polypeptide P88, which was shown to be the precursor of the capsid polypeptides, is translated from the gene located at the 5'-end of the genome. The order of the structural polypeptides, determined by the use of emetine, is VP4, VP2, VP3, VP1. The order of the remaining primary cleavage products is P52, P56 and P100. P56 is a stable product, identical with the virus infection associated (VIA) antigen found in virus harvests. The function of the other two products P52 and P100 is not known. EMDV thus differs from other picornaviruses in that there is an extra primary cleavage product, apparently resulting from translation of more of the virus genome.

Aphthovirus↗
PubMed1976

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.

Animals↗
PubMed1975

A comparative chemical and serological study of the full and empty particles of foot-and mouth disease virus.

The chemical and serological properties of the full, naturally occurring empty and artificially produced empty particles of foot-and-mouth disease virus, serotype A(subtype 10, strain 16) have been studies. The full 146S particles comprised the virus RNA, three polypeptides (VP1 to VP3) mol. wt. about 30 X 10-3, one polypeptide (VP4) mol. wt. about 13-5 X 10-3, and a small amount of a polypeptide (VPo) mol. wt. about 43 X 10-3. The naturally occurring 75S empty particles contained no RNA and much less VP1 and VP4 than were found in the fall particles. However they contained a much greater proportion of VPo than the full particles. Dialysis of purified full particles against tris-EDTA, pH 7-6, produced artificial 75S empty particles which contained only a small amount of RNA and no VP4; otherwise the polypeptide composition was similar to that of the full particles. Immunological and serological tests showed that the full particles were antigenically similar to the naturally occurring empty particles but distinct from the artificial empty particles. The latter particles, however, had serological properties similar to those of the 12S protein subunit of the virus. Both the full and naturally occurring empty particles attached efficiently to susceptible cells, whereas the artificial empty particles attached only to a limited extent. The results are related to the function of the individual polypeptides of the virus particle and compared with published work on other picornaviruses.

Animals↗
PubMed1975

A high density component in several vertebrate enteroviruses.

In addition to the major infective component, which bands at a density of 1:34 g/ml in caesium chloride ("light component"), a component with a density of 1:44 g/ml ("heavy component") has been found in harvests of poliovirus (type I), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1 . 34 g/ml but approx. 2% was present as a peak at 1 . 44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.

Capsid↗