PubMed Health⌕ Search

Biomedical subjects

S G Siddell

Publications and source records attributed to S G Siddell.

At least 19 recordsLinked to original sources
PubMed2005

Reverse genetics of coronaviruses using vaccinia virus vectors.

In this article, we describe the reverse genetic system that is based on the use of vaccinia virus cloning vectors. This system represents a generic approach to coronavirus reverse genetics and was first described for the generation of recombinant human coronavirus 229E representing a group I coronavirus. Subsequently, the same approach has been used to generate recombinant avian infectious bronchitis coronavirus and, recently, recombinant mouse hepatitis virus, representing group III and group II coronaviruses, respectively. We describe how vaccinia virus-mediated homologous recombination can be used to introduce specific mutations into the coronavirus genomic cDNA during its propagation in vaccinia virus and how recombinant coronaviruses can be isolated. Finally, we describe how the coronavirus reverse genetic system has now been extended to the generation of coronavirus replicon RNAs.

Cloning, Molecular↗
PubMed2001

Viral replicase gene products suffice for coronavirus discontinuous transcription.

We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5' and 3' ends of the human coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of coronavirus-mediated transcription. These data show that the coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.

Coronavirus↗
PubMed2001

Reverse genetics system for the avian coronavirus infectious bronchitis virus.

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

Animals↗
PubMed2001

Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis.

Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine beta-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.

Animals↗
PubMed2000

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

Adenosine Triphosphatases↗
PubMed1999

A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold .

A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.

Amino Acid Sequence↗
PubMed1999

Insertion of a foreign gene into the beta-casein locus by Cre-mediated site-specific recombination.

The expression of foreign genes in transgenic animals is generally unpredictable as transgenes are integrated at random after pro-nuclear injection into fertilized oocytes. In many cases, transgene expression is inhibited by neighbouring chromatin structures or by the repeated nature of the multiple transgene copies present at the integration site. A strategy involving homologous and site-specific recombination has been devised by which single copies of a foreign gene can be inserted specifically into the locus of a highly expressed gene. As a first step, a loxP recombination target site is introduced by homologous recombination into a predetermined gene locus such that the loxP sequence is placed next to the promoter region and replaces the translational initiation signal. In a subsequent site-specific recombination reaction, a gene of interest can be integrated into the pre-existing loxP site. This biphasic recombination strategy was used to integrate a luciferase reporter gene into the locus of the murine beta-casein gene in embryonic stem cells.

Animals↗
PubMed1999

Processing of the human coronavirus 229E replicase polyproteins by the virus-encoded 3C-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab.

Replicase gene expression by the human coronavirus 229E involves the synthesis of two large polyproteins, pp1a and pp1ab. Experimental evidence suggests that these precursor molecules are subject to extensive proteolytic processing. In this study, we show that a chymotrypsin-like enzyme, the virus-encoded 3C-like proteinase (3CLpro), cleaves within a common region of pp1a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kDa are processed from pp1a/pp1ab by proteolysis of the peptide bonds Q3546/S3547, Q3629/S3630, Q3824/N3825, and Q3933/A3934. Relative rate constants for the 3CLpro-mediated cleavages Q2965/A2966, Q3267/S3268, Q3824/N3825, and Q3933/A3934 were derived by competition experiments using synthetic peptides and recombinant 3CLpro. The results indicate that coronavirus cleavage sites differ significantly with regard to their susceptibilities to proteolysis by 3CLpro. Finally, immunoprecipitation with specific rabbit antisera was used to detect the pp1a/pp1ab processing end products in virus-infected cells, and immunofluorescence data that suggest an association of these polypeptides with intracellular membranes were obtained.

Amino Acid Sequence↗
PubMed1998

Replication and transcription of HCV 229E replicons. p6.

Replicons based upon the human coronavirus 229E (HCV 229E) genome were transfected into HCV 229E infected cells. We demonstrate that a synthetic RNA comprised of 646 nucloetides from the 5' end and 1465 nucloetides from the 3' end of the HCV 229E genome is replication competent. We conclude that the cis-acting elements necessary for replication are located in these 5' and 3' genomic regions. Furthermore, we inserted the intergenic region of the HCV 229E nucleocapsid protein gene into this basic construct and were able to demonstrate the transcription of "subgenomic" RNAs.

Coronavirus↗
PubMed1998

Substrate specificity of the human coronavirus 229E 3C-like proteinase.

Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyproteins and a key enzyme in this process is the virus-encoded 3C-like proteinase. In this study, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the substrate specificity of the purified protein. Using immunofluorescence microscopy, we have also investigated the subcellular localization of the 3C-like proteinase and have found a punctate, perinuclear distribution of the proteinase in virus-infected cells.

3C Viral Proteases↗
PubMed1998

Characterization of a papain-like cysteine-proteinase encoded by gene 1 of the human coronavirus HCV 229E.

Expression of the coronaviral gene 1 polyproteins, pp 1a and pp 1ab, involves a series of proteolytic events that are mediated by virus-encoded proteinases similar to cellular papain-like cysteine-proteinases and the 3C-like proteinases of picornaviruses. In this study, we have characterized, in vitro, the human coronavirus HCV 229E papain-like cysteine-proteinase PCP 1. We show that PCP 1 is able to mediate cleavage of an aminoterminal polypeptide, p9, from in vitro translation products representing the aminoproximal region of pp 1a/pp 1ab. Mutagenesis studies support the prediction of Cys1054 and His1278 as the catalytic amino acids of the HCV 229E PCP 1, since mutation of these residues abolishes the proteolytic activity of the enzyme.

Coronavirus↗
PubMed1998

Long distance RT-PCRs of human coronavirus 229E RNA.

The generation and cloning of cDNA fragments longer than 10 kb is often a difficult and time consuming task. In this study, we have analysed the conditions necessary of produce reverse transcripts longer than 10 kb that can be amplified by polymerase chain reaction. Thus, we isolated poly(A)-RNA from human coronavirus 229E infected MRC-5 cells and did reverse transcription using a sequence-specific primer. Subsequently, we amplified PCR products of varying length upstream of the primer position. Optimisation of the poly(A)-RNA preparation, the reverse transcription protocol and the polymerase chain reaction cycle conditions enabled us to successfully amplify regions of the human coronavirus 229E genome between 11.5 and 20.3 kb in length.

Cell Line↗
PubMed1998

Molecular analysis of the coronavirus-receptor function of aminopeptidase N.

Aminopeptidase N (APN) is a major cell surface for coronaviruses of the serogroup I. By using chimeric APN proteins assembled from human, porcine and feline APN we have identified determinants which are critically involved in the coronavirus-APN interaction. Our results indicate that human coronavirus 229E (HCV 229E) is distinct from the other serogroup I coronaviruses in that determinants located within the N-terminal parts of the human and feline APN proteins mediate the infection of HCV 229E, whereas determinants located within the C-terminal parts of porcine, feline and canine APN mediate the infection of transmissible gastro-enteritis virus (TGEV), feline infectious peritonitis virus (FIPV) and canine coronavirus (CCV), respectively. A further analysis of the mapped amino acid segments by site directed mutagenesis revealed that a short stretch of 8 amino acids in the hAPN protein plays a decisive role in mediating HCV 229E reception.

Amino Acid Sequence↗
PubMed1998

Isolation and recombinant expression of an MHV-JHM neutralising monoclonal antibody.

The monoclonal antibody A1 (mab A1) efficiently neutralises the infection of susceptible cells by the murine hepatitis virus MHV-JHM in vitro and in vivo (Wege et al., 1984). The variable regions of mab A1 were amplified from mRNA of the respective hybridoma cell line by RT-PCR and integrated into different eukaryotic expression vectors. The biological function of the recombinant antibody constructs was verified by virus neutralisation assays. Whereas a complete recombinant antibody (mab A1rec.) expressed in transfected murine myeloma cells inhibited the MHV-JHM infection as well as the parental antibody, a single-chain Fv derived from mab A1 did not show any neutralising activity.

Animals↗
PubMed1998

Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate.

Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitro trans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus ppla and pplab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861-Glu-975 and Asn-1209-Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.

Amino Acid Sequence↗
PubMed1997

Genomic targeting of a bicistronic DNA fragment by Cre-mediated site-specific recombination.

The Cre-recombinase of bacteriophage P1 catalyses site-specific recombination between DNA fragments containing loxP sites. Targeting of predefined genomic loci can be achieved by Cre-mediated linkage of a promoterless resistance marker gene to a floxed promoter pre-existing in the genome. In order to avoid the introduction of plasmid sequences into the host genome, we have constructed a series of plasmids in which the DNA segment to be integrated is flanked by two loxP sites. We show here that this floxed targeting fragment is reliably and effectively separated from the vector backbone and integrated into genomic loxP sites by Cre-mediated site-specific recombination in mammalian cells. We also demonstrate that by using this approach two convergent, promoterless coding regions can simultaneously be linked to two independent promoter elements at a pre-existing genomic loxP site. This methodology will be particularly useful for genomic targeting experiments in transgenic animals.

Animals↗