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T B Macnaughton

Publications and source records attributed to T B Macnaughton.

17 recordsLinked to original sources
PubMed2001

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes.

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

Animals↗
PubMed2000

RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases.

Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of alpha-amanitin (<3 microgram/ml) and could be partially restored by an alpha-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by alpha-amanitin to a concentration higher than 25 microgram/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

Animals↗
PubMed1999

A cellular protein which binds hepatitis B virus but not hepatitis B surface antigen.

The envelope of hepatitis B virus (HBV) consists of three related proteins known as the large (L), middle (M) and small (S) hepatitis B surface antigens (HBsAg). L-HBsAg has a 108-119 amino acid extension at the N terminus compared with M-HBsAg and contains the preS1 sequence of the HBV envelope. Previous research has identified this region as the likely virus attachment protein which is thought to interact with the cellular receptor for the virus. However, as the receptor has still not been identified unequivocally, we used the preS1 region of L-HBsAg to screen a human liver cDNA library by the yeast two-hybrid system. Several positive clones were isolated which encoded cellular proteins that interacted with the HBV preS1 protein. The specificity was examined in an independent manner in experiments in which baculovirus-derived glutathione S-transferase (GST)-preS1 was incubated with 35S-labelled protein expressed by in vitro translation from the positive clones. The intensity of the interactions using this alternative approach mirrored those observed in the yeast two-hybrid system and two proteins (an unidentified protein and a mitochondrial protein) were selected for further study. The specificity of the binding reaction between the preS1 protein and these two proteins was further confirmed in a competition assay; HBV purified from serum, but not purified HBsAg, was able to compete with preS1 and thus block GST-preS1 binding to the unidentified protein but not to the mitochondrial protein. The unidentified protein was then expressed as a fusion protein with GST and this was able to bind HBV virions in a direct manner.

Baculoviridae↗
PubMed1997

The development and characterisation of a SV40 T-antigen positive cell line of human hepatic origin.

COS7 cells and other cell lines expressing the SV40 large T-antigen, have been of great benefit in transfection studies using transient expression vectors which contain the SV40 origin of replication, as these cells allow plasmid replication to a high copy number. As no T-antigen expressing cell line derived from a well characterised hepatocyte-like continuous cell line currently exists, the establishment of such a cell line for studies which require expression of hepatocyte-specific factors would be extremely useful. A HepG2-derived stable cell line (THT1) was therefore developed which demonstrates a high level of transfection efficiency whilst retaining hepatocyte-like features, such as the production of hepatitis B virus. The THT1 cell line displayed chromosomal integration of the SV40 T-antigen gene and nuclear expression of the antigen. The cell line also maintained the general morphological features of its parent cell line, and showed an increased rate of cell growth.

Animals↗
PubMed1996

Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhoea virus.

The noncytopathic Australian bovine viral diarrhoea virus (BVDV) Trangie isolate was used to establish a one-step growth curve and to investigate previously uncharacterized aspects of pestivirus replication. The eclipse phase was found to be approximately 8 to 10 h and the first appearance of viral antigen assayed by immunofluorescence occurred around 6 h post-infection (p.i.). Both positive- and negative-sense virus RNAs were first detected at 4 h p.i. by Northern blot hybridization using strand-specific RNA probes generated by in vitro transcription from cDNA cloned from the NS3 region. The ratio of positive- to negative-sense virus RNA changed from 2:1 at 4 h p.i. to 10:1 at 12 h p.i. and thereafter. The kinetics of synthesis showed that the rate of synthesis of positive-strand viral RNA increased rapidly from 6 h p.i., whereas the rate of synthesis of the negative-strand remained constant. The copy number of genomic RNA determined by Northern blot hybridization analysis was estimated to be 1.5 x 10(4) copies per cell, 16 to 24 h p.i. Viral RNA species that were thought to represent replicative intermediate (RI) and replicative forms (RF) were detected after electrophoretic separation by urea-PAGE. Confirmation of the identity of the RI and RF was obtained using LiCl precipitation and RNase A digestion of [3H]uridine-labelled RNA. Pulse-chase labelling of BVDV-infected cells was consistent with synthesis of nascent BVDV RNA through an RI derived by strand displacement from an RF template and thus the synthesis of BVDV RNA is likely to be similar to the model proposed for flavivirus replication.

Animals↗
PubMed1994

Adsorption and penetration of hepatitis B virus in a nonpermissive cell line.

A cell monolayer radioimmunoassay was established to detect hepatitis B virus (HBV)-binding activity. HBV was shown to bind to HepG2 and HuH7 cells, but not to LTK- and HeLa cells. The binding activity was inhibited by peptide 21-47 from the large hepatitis B surface antigen (L-HBsAg) region, but not by other peptides from the L- and middle-HBsAg. A monoclonal antibody to L-HBsAg (MA18/7) and a polyclonal antibody to HBsAg (anti-HBs) also inhibited the binding. However, only the Fab fragment of MA18/7 showed blocking activity, suggesting that these two antibodies may neutralize virus by different mechanisms. Excess HBV was able to saturate the binding activity of HepG2 cells. The virus was also shown to be internalized; virus DNA was detected in the cytoplasmic fraction 1-2 hr postadsorption and was associated with the nuclear fraction 2 hr later. Furthermore, immunoprecipitation experiments showed that the virus DNA remained encapsidated 48 hr after internalization. Nuclear fractionation experiments showed that the encapsidated HBV DNA remained associated with the nuclear membrane. Trypsinization of intact nuclei resulted in disassociation of the nucleocapsid from the nuclear membranes, suggesting that the nuclear membrane presents a barrier to intact HBV virions or nucleocapsids. This may explain why HepG2 cells are refractile to infection although permissive for HBV replication after transfection of viral DNA.

Antibodies, Monoclonal↗
PubMed1993

Natural evolution of coronavirus defective-interfering RNA involves RNA recombination.

Defective-interfering (DI) RNAs of RNA viruses, in general, are generated and continue to evolve in size during serial undiluted passages of viruses. This evolution was thought to occur by independent generation of DI RNAs during virus passages and subsequent selection of new DI RNAs under new cellular conditions. Here we demonstrate that recombination between the old DI RNA and the helper viral RNA can be one of the mechanisms for natural DI RNA evolution. A mouse hepatitis virus (MHV) DI RNA, DIssE RNA, was transcribed in vitro and transfected into a mouse cell line infected with a different MHV strain (A59), which is distinguishable from the original natural helper MHV (JHM). During subsequent serial undiluted passages of the harvested virus, several novel DI RNA species were generated, while the original DIssE RNA disappeared by passage 11. cDNA cloning and sequence analysis of one of these novel DI RNAs, designated DI-2, revealed that it is composed of four discontinuous regions of the genomic sequence and is different from the structure of the original DIssE RNA. Sequence comparison among DI-2, DIssE, and helper MHV-A59 RNAs showed that DI-2 sequence is similar to DIssE in the first and second regions, but similar to the helper A59 virus in the third and last regions. Thus, this DI RNA was generated by RNA recombination between the original DIssE RNA and the helper viral RNA. These results indicate that recombination between DI RNA and helper virus RNA can be involved in the natural evolution of DI RNAs.

Animals↗
PubMed1993

Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template.

Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA, they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.

Base Sequence↗
PubMed1993

Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites.

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.

Animals↗
PubMed1991

Direct evidence for cytotoxicity associated with expression of hepatitis delta virus antigen.

It has been postulated that hepatocyte injury resulting from infection with hepatitis D virus may be caused by a direct virus cytotoxicity in contrast to immune-mediated injury associated with hepatitis B virus. We have transfected HeLa and HepG2 continuous cell lines with a recombinant plasmid containing the hepatitis D antigen gene under the inducible control of the human metallothionein promoter. The addition of zinc to the cell culture medium then led to the expression of hepatitis D antigen associated with, in the short term, a significant reduction in the rate of RNA but not DNA synthesis and, in the longer term, cell death. The necrotic cells had pyknotic nuclei and shrunken eosinophilic cytoplasm; these necrotic cells resembled the apoptotic bodies seen in hepatitis D virus-related hepatitis. The level of hepatitis D antigen in individual cells that produced these changes was similar to the level of hepatitis D antigen in hepatocytes from a chimpanzee with acute hepatitis D virus infection. We conclude that the expression of hepatitis D antigen resulted in significant cytotoxic changes in these cells, providing strong support for the view that hepatitis D antigen may be specifically cytotoxic to infected hepatocytes in vivo.

Animals↗
PubMed1991

Cell culture model systems to study HDV expression, replication and pathogenesis.

Due to the transient nature of HDV replication in natural and experimental hosts and to the inability of the virus to replicate in easily handled cell lines, studies of the replication and expression of HDV have been impeded. These difficulties have been overcome by the development of the cell lines described in this paper.

Animals↗
PubMed1990

Hepatitis delta virus RNA, protein synthesis and associated cytotoxicity in a stably transfected cell line.

A trimer of hepatitis delta virus (HDV) cDNA in a retrovirus expression vector was transfected into subclone of the PLC/PRF/5 human hepatoma cell line, and a stable cell line (H1 delta 9) was clonally selected that supported the synthesis of both genomic and antigenomic sense HDV RNA. The H1 delta 9 cell line also expressed hepatitis delta antigen (HDAg) in cell nuclei in three distinct morphological patterns, including patterns typically seen in HDV-infected livers. HDAg expression was restricted to the smaller (p24) of the two HDAg-associated polypeptides in early passages of the H1 delta 9 cell line, but continuous passage of the cells resulted in increasing of expression of the larger (p27) HDAg-specific polypeptide. Passage of the H1 delta 9 cells also led to sustained expression of monomeric HDV RNA and a reduction in the levels of dimeric- and trimeric-HDV RNA. This was accompanied by an attenuation of virus-related cytotoxicity which was a feature of early cell passage numbers. HDV RNA replication in these cells was resistant to actinomycin D suggesting that replication was not dependent on continued expression from the transfected HDV cDNA and thus was likely to be self-sustaining.

Blotting, Northern↗
PubMed1990

Use of recombinant hepatitis delta antigen in diagnostic assays for HDV antibody.

The gene encoding the hepatitis delta virus (HDV) structural antigen (HD Ag) was inserted into a Rous sarcoma virus expression vector and the recombinant plasmid used to direct the synthesis of recombinant HD Ag (rHD Ag) in a continuous hepatoma cell line. A competitive radioimmunoassay for serum antibody to HDV using rHD Ag was developed and was found to be equally suitable for diagnostic purposes to a radioimmunoassay using infected liver-derived HD Ag. Similarly, rHD Ag was shown to be serologically equivalent to liver-derived HD Ag within the limit of the blocking titrations performed. The rHD Ag-positive cell line was also used in an indirect immunofluorescence assay to detect anti-HD. Similar titres of anti-HD were detected by both radioimmunoassay and immunofluorescence and identical samples were positive for anti-HD by either assay. In a sample of prison inmates with high prevalence of both HBV and HDV, anti-HD was confined almost exclusively to those with persistent HBV infection and not to those in whom HBV infection had cleared. The availability of rHD Ag will permit wider development of diagnostic anti-HD assays, and the use of two such assays is presented in this study.

Antigens, Viral↗
PubMed1990

Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

Antigens, Viral↗