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Biomedical subjects

D V Sangar

Publications and source records attributed to D V Sangar.

At least 19 recordsLinked to original sources
PubMed2000

Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3.

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.

Amino Acid Sequence↗
PubMed2000

Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.

The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

Chloramphenicol O-Acetyltransferase↗
PubMed1999

An infectious molecular clone of a Japanese genotype 1b hepatitis C virus.

We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.

3' Untranslated Regions↗
PubMed1998

Examination of poliovaccines for the presence of SV40 sequences.

Batches of monovalent oral poliovirus vaccine bulks previously screened for infectious SV40 by cell culture methods and used in the United Kingdom from 1971-96 in trivalent oral poliovirus vaccine have now been examined by a newly developed PCR for the presence of SV40 sequences. Up to April 1997, over 190 batches have been examined. SV40 sequences were not detected in any of the vaccines. This provides additional assurance that oral poliovirus vaccines used in the U.K. from 1971 are free of SV40 contamination.

Animals↗
PubMed1995

In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease.

The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.

Adenosine Triphosphatases↗
PubMed1994

Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro.

The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.

Animals↗
PubMed1993

The nature and spatial distribution of amino acid substitutions conferring resistance to neutralizing monoclonal antibodies in human rhinovirus type 2.

A total of 38 neutralization escape mutant viruses have been selected from a cloned stock of human rhinovirus serotype 2 (HRV-2), using either of two monoclonal antibodies (MAbs) which recognize overlapping epitopes as judged by competition binding. The mutant viruses were analysed for their sensitivity to a panel of antiviral MAbs by antibody binding and virus neutralization assays. The position and nature of the selected mutations was determined by sequencing of the virus RNAs, and the location of the substituted amino acids on the three-dimensional structure of the virus predicted from the co-ordinates determined for the closely related HRV-1A. Escape from neutralization could be attributed to single amino acid substitutions in all but one case, which had a deletion of four amino acids. In all cases in which the same mutation was found more than once, these mutations were transitions. The ratio of transition to transversion mutations was about 5:1 overall or about 1.7:1 if only unique substitutions are considered. Each antibody selected for a discrete cluster of mutations and the area of these clusters was considerably less than that determined to be in contact with antibodies from X-ray crystallographic analyses of antibody/protein complexes. One mutation did not occur within the cluster of others selected with the same antibody. This substitution occurred at the base of a small loop and may cause conformational changes at the virus surface.

Amino Acids↗
PubMed1991

Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines.

We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines. Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency. Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results. The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 degrees C and 37 degrees C. The maximum level of attachment was observed at 4 degrees C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays. The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting. The data show that such components are of high Mr and that the serum glycoprotein, alpha 2-macroglobulin, can partly mimic the inhibitory effect of whole serum.

Adsorption↗
PubMed1988

Processing and assembly of foot-and-mouth disease virus proteins using subgenomic RNA.

Recombinant DNA clones were constructed in order to study the mechanisms of proteolytic processing and assembly in foot-and-mouth disease virus (FMDV). RNA transcripts from these clones were synthesized using SP6 polymerase and translated in rabbit reticulocyte lysates. Efficient translation occurred in the absence of all 5' untranslated sequences and processing of the structural proteins occurred in the presence of functional 3C protease which can function in trans. The specificity of 3C protease activity is not limited to Glu-Gly bonds. Translation of correctly processed structural proteins leads to assembly of subviral structures resembling 'empty' particles. Further studies on the processing of the FMDV genome show that the primary cleavage (P1-P2) is mediated neither by 3C nor the second FMDV protease L. Preliminary evidence suggests that an initial very rapid cleavage occurs between 2A and 2B with subsequent cleavage of the P1/2A junction probably being carried out by 3C.

Amino Acid Sequence↗
PubMed1988

Modification of the leader protein (Lb) of foot-and-mouth disease virus.

Translation of foot-and-mouth disease virus RNA for extended periods in rabbit reticulocyte lysates results in the appearance of a previously undescribed protein. A protein with similar properties can also be detected in BHK cells at late times after virus infection. Specific immunoprecipitation has shown that this protein (Lb') is closely related to the smaller of the two leader proteins, Lb, although it migrates with an apparently higher Mr in SDS-polyacrylamide gels. The conversion of Lb to Lb' can be mimicked by treatment with carboxypeptidase B. It is suggested that C-terminal trimming of Lb to produce Lb' results in an increase in negative charge and is responsible for its slower migration in SDS-PAGE.

Aphthovirus↗
PubMed1987

All foot and mouth disease virus serotypes initiate protein synthesis at two separate AUGs.

Translation of the foot and mouth disease virus genome in vitro and in vivo indicated that all seven serotypes initiate protein synthesis at two separate AUGs. Sequence analysis of the region surrounding these AUGs has shown that the efficiency with which the initiating AUG is recognized is dependent on the flanking nucleotides. However, in vitro, the major factor determining which AUG is used is the concentration of Mg2+.

Aphthovirus↗
PubMed1987

A synthetic peptide which elicits neutralizing antibody against human rhinovirus type 2.

Synthetic peptides corresponding to six predicted immunogenic sites on human rhinovirus type 2 (HRV2) have been tested for their reactivity with an anti-virion antibody and for their ability to elicit neutralizing antibody. Four of the peptides reacted with HRV2 antiserum in an indirect ELISA. Rabbit antisera produced to three of these four peptides, one each from VP1, VP2 and VP3, reacted with the virus in an indirect ELISA and with the corresponding proteins by Western blotting. Furthermore, antiserum to one of the peptides, designed to cover the neutralization epitope NIm-II on VP2, not only reacted well in a sandwich ELISA and in an immunoprecipitation test but also neutralized virus infectivity.

Animals↗
PubMed1986

An electroblotting technique for assessing the integrity of the major immunogenic protein in foot-and-mouth disease virus vaccines.

A method has been developed whereby VP1, the major immunogenic protein of foot-and-mouth disease virus can be detected after electroblotting on nitrocellulose paper. Proteins can be examined in unfractionated virus harvests and after formulation as aluminium hydroxide-adjuvanted vaccines. The limit of detection is approximately 10 ng of VP1 and up to 20 samples can be analysed simultaneously. The technique allows the integrity of VP1 to be examined in fully formulated vaccines.

Animals↗
PubMed1986

The genome-linked proteins of aphthoviruses: specific immunoprecipitation of the three species detected on virus RNA and identification of possible precursors.

Synthetic peptides have been made corresponding to the C-terminal portion of each of the three presumptive genome-linked proteins (VPgs) of foot-and-mouth disease virus type A10. Antisera against each of these peptides efficiently precipitated only the homologous VPg, and the reactions were inhibited by prior absorption with homologous, but not heterologous synthetic peptide. The peptide antisera precipitated a number of proteins from infected cell extracts with mol. wt. of 100, 84, 56, 36, 27, 25 and 20, all X 10(3); all these reactions were inhibited by absorption with homologous peptide, indicating that they were probable precursors of VPg. The relationship between these proteins is at present unclear.

Amino Acid Sequence↗
PubMed1985

Analysis of the terminal sequences of the genome segments of four orbiviruses.

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.

Animals↗
PubMed1985

Two initiation sites for foot-and-mouth disease virus polyprotein in vivo.

Typically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P16, are translated from the 5' end of the coding region of the genome of foot-and-mouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (A10 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported.

Aphthovirus↗
PubMed1985

Analysis of the terminal sequences of the genome segments of four orbiviruses.

The RNA sequences of the terminal regions of the genome segments of 4 different orbiviruses were analysed. In 3 of these [bluetongue virus (BTV) types 1 and 20 and Ibaraki virus, a member of the epizootic hemorrhagic disease (EHD) serogroup], 1 strand of all of the genome segments analysed ends in 3'CAAUUU...5' while the other strand ends in 3'CAUUCA...5'. These conserved sequences are identical to those reported for BTV types 10 and 11. The 3' terminal sequences of segments 3 and 10 of the BTV 1 genome (including the conserved regions) were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all of the genome segments of these viruses) that the dsRNA molecules are fully base-paired end to end. Using in vitro synthesised mRNA of BTV 1 in annealing experiments with the separated ssRNAs from the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA...5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU...5' is of the opposite (-ve) polarity. The 4th virus analysed (Tilligerry, a member of the Eubenangee serogroup) only had 5 conserved bases at the 3' termini of 1 strand of each genome segment (3'CAU-CA...5') and 3 conserved bases at the 3' termini of the other strand (3'CA--U...5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments, particularly between the different BTV types. There was little evidence however for absolute conservation of "segment specific" sequences in these regions of the RNA, as reported for rotaviruses. The percentage of sequence homology found in the terminal regions of the genome segments between members of the BTV, EHD and Eubenangee serogroups examined correlates with the serological interrelationships between these groups of viruses. The conserved terminal sequences and sequence homology in these regions of the RNA are discussed as to their role in virus replication and their significance to the reassortment of genome segments between different viruses during dual infections.

Base Sequence↗