Biomedical subjects

S G Siddell

Publications and source records attributed to S G Siddell.

At least 37 records · Page 2Linked to original sources

PubMed1997

Effective amplification of 20-kb DNA by reverse transcription PCR.

Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.

Base Sequence↗

PubMed1997

Expression of a recombinant monoclonal antibody from a bicistronic mRNA.

The variable regions of the murine monoclonal antibody A1, which effectively neutralizes the infection of susceptible cells by the murine hepatitis virus strain JHM, were cloned, sequenced, and expressed in mammalian cells as a functional recombinant antibody. To accomplish the concurrent synthesis of both antibody chains, the light- and heavy-chain-coding regions were inserted into a bicistronic expression cassette based on the encephalomyocarditis virus internal ribosomal entry site. The strategy of combining both coding regions in one bicistronic mRNA allows for the rapid isolation of cell clones expressing high levels of recombinant antibody.

Amino Acid Sequence↗

PubMed1997

Expression and characterization of a recombinant murine coronavirus 3C-like proteinase.

The replication of coronaviruses involves proteolytic processing of the gene 1 translation products, pp1a and pp1ab. One of the key enzymes in this process is predicted to be a virus-encoded 3C-like proteinase. In this report, we describe a bacterial system that has allowed us to express and characterize a recombinant murine coronavirus (MHV-JHM) 3C-like proteinase. The partially purified protein has been shown to exhibit proteolytic activity in trans and mutation analysis has been used to demonstrate the indispensability of Cys-3495 for enzymatic activity. Finally, the effect of class-specific proteinase inhibitors on the trans cleavage activity of the MHV 3C-like proteinase has been used to demonstrate the functional and structural homology of this enzyme to the picornavirus 3C proteinases.

Amino Acid Sequence↗

PubMed1997

Identification and subcellular localization of a 41 kDa, polyprotein 1ab processing product in human coronavirus 229E-infected cells.

The translation products of the human coronavirus (HCV) 229E open reading frames 1a and 1b, the polyproteins 1a and 1ab, are processed by virus-encoded proteinases. One of the key enzymes in this process is a chymotrypsin-like enzyme, the 3C-like proteinase. In this study we have identified an ORF 1b-encoded, 41 kDa processing product in HCV 229E-infected cells by using a monoclonal antibody with defined specificity. We show that this polypeptide is released from polyprotein 1ab by 3C-like proteinase-mediated cleavage of the peptide bonds Gln-6110/Gly-6111 and Gln-6458/Ser-6459. Also, we have investigated the subcellular localization of the 41 kDa processing product. Immunofluorescence microscopy revealed a punctate, perinuclear distribution of the 41 kDa polypeptide in infected cells and an identical subcellular localization was observed for three additional pp1ab-derived polypeptides. In contrast, the virus nucleocapsid protein showed a homogeneous cytoplasmic localization.

Antibodies, Monoclonal↗

PubMed1997

Identification of residues critical for the human coronavirus 229E receptor function of human aminopeptidase N.

Aminopeptidase N (APN) is the major cell surface receptor for group 1 coronaviruses. In this study, we have isolated and characterized a feline APN cDNA and shown that the transfection of human embryonic kidney cells with this cDNA renders them susceptible to infection with the feline coronavirus feline infectious peritonitis virus, the human coronavirus (HCV) 229E and the porcine coronavirus porcine transmissible gastroenteritis virus. By using chimeric APN genes, assembled from porcine and feline sequences, we have shown that, analogously to the human APN protein, a region within the amino-terminal part of the feline APN protein (encompassing amino acids 132-295) is essential for its HCV 229E receptor function. Furthermore, by comparing the relevant feline, human and porcine APN sequences, we were able to identify a hypervariable stretch of eight amino acids that are more closely related in the feline and human APN proteins than in the porcine APN molecule. Using PCR-directed mutagenesis, we converted this stretch of amino acids within the porcine APN molecule to the corresponding residues of the human APN molecule. These changes were sufficient to convert porcine APN into a functional receptor for HCV 229E and thus define a small number of residues that are critically important for the HCV 229E receptor function of human APN.

Amino Acid Sequence↗

PubMed1997

Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase.

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase. In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein. Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate. Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity. Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity. These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties.

Amino Acid Sequence↗

PubMed1997

Identification of an ATPase activity associated with a 71-kilodalton polypeptide encoded in gene 1 of the human coronavirus 229E.

Human coronavirus 229E gene expression involves proteolytic processing of the gene 1-encoded polyproteins pp1a and pp1ab. In this study, we have detected a 71-kDa polypeptide in virus-infected cells that is released from pp1ab by the virus-encoded 3C-like proteinase and that has been predicted to contain both metal-binding and helicase domains. The polypeptide encompasses amino acids Ala-4996 to Gln-5592 of pp1ab and exhibits nucleic acid-stimulated ATPase activity when expressed as a fusion protein with the Escherichia coli maltose-binding protein. These data provide the first identification of a coronavirus open reading frame 1b-encoded enzymatic activity.

3C Viral Proteases↗

PubMed1996

Genomic targeting with an MBP-Cre fusion protein.

The Cre recombinase of bacteriophage P1 catalyses site-specific recombination between lox-recombination target sites both in prokaryotic and eukaryotic cells and has thus become a popular tool in genetic research. Stable, Cre-mediated integration of DNA sequences at pre-existing lox sites in the eukaryotic genome is facilitated when a Cre recombinase protein rather than a cre-expression plasmid is used to direct site-specific recombination (Baubonis and Sauer (1993) Nucleic Acids Res., 21, 2025-2029). We bacterially produced a Cre recombinase containing a nuclear localisation signal as a fusion protein with the E. coli maltose binding protein (MBP) and purified the protein by one step affinity chromatography. Subsequent cleavage with the protease factor Xa releases the Cre recombinase including the nuclear localisation signal from the maltose binding protein. Surprisingly, we found that the recombination activity of the uncleaved MBP-Cre fusion protein is virtually identical to that of the native Cre recombinase. This suggests that the MBP portion of the fusion protein behaves as a separate protein domain which does not interfere with Cre activity and can thus be used as an independent molecular tag. Additionally, the fusion protein is very resistant to proteolytic degradation and active over a wide range of temperatures. It efficiently catalyses excision and integration reactions in vitro and in eukaryotic cells. Finally, we could show that, by using MBP-Cre, it is possible to concomitantly excise a lox-flanked DNA sequence from a plasmid and integrate it into a pre-existing lox site in the genome in one transfection experiment. Vector backbone sequences which might have undesirable effects can thereby be excluded. The MBP-Cre fusion protein described here will be a useful tool not only for the catalysis of Cre-mediated recombination reactions in vitro and in vivo but also for the analysis of the mechanism of site-specific recombination.

ATP-Binding Cassette Transporters↗

PubMed1996

Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E.

Gene 1 of the human coronavirus HCV 229E encompasses approximately 20.7 kb and contains two overlapping open reading frames, ORF 1a and ORF 1b. The downstream ORF 1b is expressed by a mechanism involving (-1) ribosomal frameshifting. Translation of mRNA 1, which is thought to be equivalent to the viral genomic RNA, results in the synthesis of two large polyproteins, pp1a and pp1ab. These polyproteins contain motifs characteristic of papain-like and 3C-like proteinases, RNA-dependent RNA polymerases, helicases, and metal-binding proteins. In this study, we have produced pp1ab-specific monoclonal antibodies and have used them to detect an intracellular, 105-kDa viral polypeptide that contains the putative RNA polymerase domain. Furthermore, using trans cleavage assays with bacterially expressed HCV 229E 3C-like proteinase, we have demonstrated that the 105-kDa polypeptide is released from pp1ab by cleavage at the dipeptide bonds Gln-4068/Ser-4069 and Gln-4995/Ala-4996. These data contribute to the characterization of coronavirus 3C-like proteinase-mediated processing of pp1ab and provide the first identification of an HCV 229E ORF 1ab-encoded polypeptide in virus-infected cells.

3C Viral Proteases↗

PubMed1996

Characterization of functional domains in the human coronavirus HCV 229E receptor.

Human aminopeptidase N (hAPN or CD13) and porcine aminopeptidase N (pAPN) are functional receptors for human coronavirus (HCV) 229E and porcine transmissible gastroenteritis virus (TGEV), respectively. However, hAPN cannot function as a receptor for TGEV and pAPN cannot function as a receptor for HCV 229E. In this study, we constructed a series of chimeric hAPN/pAPN genes and expressed the corresponding proteins in transfected cells. Subsequently, we identified the chimeric proteins that can function as a receptor for HCV 229E. The results show that replacement of a small region of pAPN sequence (pAPN amino acids 255-348) with the corresponding hAPN sequence (hAPN amino acids 260-353) converts pAPN into a functional receptor for HCV 229E. The region of hAPN that we have defined in this way does not correspond to the region of pAPN that has been identified as essential for the TGEV-receptor interaction. We conclude that although both viruses use a homologous receptor protein, different regions of the protein are required to mediate susceptibility to infection with HCV 229E and TGEV.

Animals↗

PubMed1995

Detection of human coronavirus 229E-specific antibodies using recombinant fusion proteins.

Human coronaviruses are known to be a common cause of respiratory infections in man. However, the diagnosis of human coronavirus infections is not carried out routinely, primarily because the isolation and propagation of these viruses in tissue culture is difficult and time consuming. The aim of this study was to evaluate the use of recombinant, bacterial expressed proteins in the serodiagnosis of coronavirus infections. Two proteins were examined: the human coronavirus 229E nucleocapsid protein (N), expressed as a fusion protein in the vector pUR and the coronavirus 229E surface glycoprotein (S), expressed as a fusion protein in the vector pROS. The recombinant proteins were used as antigens in Western blot (WB) assays to detect the 229E-specific IgG antibodies and the results were compared with a standard serological method, indirect immunofluorescence. Serum samples of 51 paediatric patients, suffering from acute respiratory illness, and 10 adults, voluntarily infected with human coronavirus, were tested. The serum samples of the adult group had coronavirus-specific IgG antibodies in both test systems. In contrast, only 8/51 sera of the paediatric group were positive for coronavirus-specific IgG by both WB and IF and 20/51 sera were positive by WB, but not by IF. The overall incidence of human coronavirus infections in the paediatric age group was 55% evaluated by WB analysis and 16% evaluated by IF. This study shows that recombinant human coronavirus 229E proteins are suitable reagents for the epidemiological screening of coronavirus 229E infections.

Adult↗

PubMed1995

Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity.

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.

Amino Acid Sequence↗

PubMed1994

Single amino acid changes in the S2 subunit of the MHV surface glycoprotein confer resistance to neutralization by S1 subunit-specific monoclonal antibody.

We have isolated 10 monoclonal-antibody-resistant variants (MAR variants) of the murine hepatitis virus (MHV) by selection with a neutralizing monoclonal antibody that binds to the S1 subunit of the surface glycoprotein (MAb 11F, E. Routledge et al., J. Virol. 65, 254-262, 1991). The variant viruses escape neutralization and are able to mediate membrane fusion in the presence of high concentrations of antibody. Sequence analysis of the variant S protein genes showed that they are not mutated in the codons for the amino acids that bind MAb 11F (positions 33 to 40). Instead, they have mutations that encode single amino acid changes in the S2 subunit of the protein (positions 1109, 1110, and 1116). These amino acid substitutions are conservative. Using a T7/vaccinia virus system, we have confirmed that each of the S2 subunit substitutions is able to confer the MAR phenotype on transiently expressed S proteins. The indirect immunoprecipitation of metabolically labeled S protein from cell lysates of MHV- or MAR variant-infected cells shows that the MAR-variant S proteins no longer bind MAb 11F, although they are still bound by a large panel of monoclonal antibodies that recognize many different discontinuous and linear determinants on both the S1 and S2 subunits of the protein. These data provide new insights into the interaction between defined regions of the S1 and S2 subunits of the MHV S protein.

Amino Acid Sequence↗

PubMed1994

Internal ribosome entry in the coding region of murine hepatitis virus mRNA 5.

The unique region of murine hepatitis virus (MHV) mRNA 5 has two open reading frames. ORF 5a and ORF 5b, that encode small proteins of unknown function. In the experiments described here, we have used the in vitro translation of synthetic mRNAs to examine the expression of these ORFs. Our results show that a synthetic mRNA containing both ORFs is functionally bicistronic. More importantly, the expression of ORF 5b, but not ORF 5a, is maintained in a tricistronic mRNA containing an additional 5'-proximal ORF. Thus, in the context of the MHV mRNA 5 unique region, the initiation of protein synthesis on ORF 5b can occur independently of ribosomes that enter from the 5' end of the mRNA. We conclude that the translation of ORF 5b is mediated by the internal entry of ribosomes.

Animals↗

PubMed1993

An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA.

The RNA polymerase gene (gene 1) of the human coronavirus 229E is approximately 20 kb in length and is located at the 5' end of the positive-strand genomic RNA. The coding sequence of gene 1 is divided into two large open reading frames, ORF1a and ORF1b, that overlap by 43 nucleotides. In the region of the ORF1a/ORF1b overlap, the genomic RNA displays two elements that are known to mediate (-1) ribosomal frameshifting. These are the slippery sequence, UUUAAAC, and a 3' pseudoknot structure. By introducing site-specific mutations into synthetic mRNAs, we have analysed the predicted structure of the HCV 229E pseudoknot and shown that besides the well-known stem structures, S1 and S2, a third stem structure, S3, is required for a high frequency of frameshifting. The requirement for an S3 stem is independent of the length of loop 2.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence of the human coronavirus 229E RNA polymerase locus.

The nucleotide sequence of the human coronavirus 229E (HCV 229E) RNA polymerase gene and the 5' region of the genome has been determined. The polymerase gene is comprised of two large open reading frames, ORF1a and ORF1b, that contain 4086 and 2687 codons, respectively. ORF1b overlaps ORF1a by 43 bases in the (-1) reading frame. The in vitro translation of SP6 transcripts which include HCV 229E sequences encompassing the ORF1a/ORF1b junction show that expression of ORF1b can be mediated by ribosomal frame-shifting. The predicted translation products of ORF1a (454,200 molecular weight) and ORF1a/1b (754,200 molecular weight) have been compared to the predicted RNA polymerase gene products of infectious bronchitis virus (IBV) and murine hepatitis virus (MHV) and conserved structural features and putative functional domains have been identified. This analysis completes the nucleotide sequence of the HCV 229E genome.

Amino Acid Sequence↗

PubMed1993

Characterization of the human coronavirus 229E (HCV 229E) gene 1.

The sequence of the HCV 229E gene 1 has been determined and compared with the homologous sequences of the murine hepatitis virus and the avian infectious bronchitis virus. The coding sequence of gene 1 is 20,273 nucleotides in length. Within this coding region are two large open reading frames, ORF 1a (4,086 codons) and ORF 1b (2,687 codons) which overlap by 40 nucleotides. In the overlapping region, the genomic RNA can be folded into a pseudoknot structure, an element which is known to mediate -1 ribosomal frame-shifting in other coronaviruses. Assuming that -1 frame-shifting occurs at the HCV sequence UUUAAAC (nucleotides 12,514-12,520), the ORF 1a - ORF 1b product is predicted to be 6,758 amino acids in length. Our sequence analysis of the HCV 229E gene 1 has revealed a high degree of similarity within the ORF 1b of HCV, MHV and IBV, whereas ORF 1a is much less conserved. Elements which are believed to be necessary for specific (e.g. frame-shifting) and general (e.g. NTP-binding/helicase) transcriptional functions have been identified. This study completes the genomic sequence of HCV 229E which is 27.27 kb long and one of the largest known RNA genomes.

Base Sequence↗

PubMed1993

Characterization of a monoclonal antibody resistant variant of MHV.

A monoclonal antibody resistant (MAR) variant of MHV was isolated after infection of hybridoma cells secreting the neutralizing and fusion-inhibiting monoclonal antibody, mAb 11F. The isolated variant was able to mediate syncytia formation even in the presence of high concentrations of mAb 11F. The S gene of the variant was cloned and sequenced. There were three nucleotide exchanges in comparison to the wild-type S gene, resulting in two amino acid alterations. However, both amino acid substitutions (at positions 255 and 1116) were located outside the binding site of mAb 11F.

Amino Acid Sequence↗