Biomedical subjects

Christopher J McCormick

Publications and source records attributed to Christopher J McCormick.

5 recordsLinked to original sources

PubMed2006

A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

Cell Line, Tumor↗

PubMed2006

Tagging of NS5A expressed from a functional hepatitis C virus replicon.

Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.

Carboxyl and Carbamoyl Transferases↗

PubMed2004

Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system.

We have developed a baculovirus delivery system that enables tetracycline-regulated expression of polII-derived hepatitis C virus (HCV) transcripts in hepatocyte-derived cell lines (McCormick et al., 2002). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a neo(R)-expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5'UTR positioned -2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5' end of the transcript (5'HH) that cleaves between the ribozyme-5'UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the beta-interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.

5' Untranslated Regions↗

PubMed2002

High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors.

BACKGROUND/AIMS: Gene transfer into hepatic stellate cells (HSC) is inefficient when using plasmid-based transfection methods; viral-based systems are therefore being developed. A baculovirus system has recently been shown to be useful for expressing genes in mammalian cells. The aim of this study was to determine if baculovirus vectors can infect and express target genes in rat and human HSC and to assess potential cytotoxic and modulatory effects of infection. METHODS: A recombinant baculovirus vector (AcCALacZ) carrying the LacZ gene was used to infect HSC. beta-Galactosidase assays and electron microscopy were used to determine efficiency of infection and gene expression. Counting of trypan blue negative cells was used to assess cytotoxic/cytostatic effects of infection. Measurement of protein content of cells and alpha-smooth muscle actin expression were performed to assess the effects of baculovirus on cell function/phenotype. RESULTS: Baculovirus infection of activated HSC was highly efficient (> 90%) and provided long-term LacZ gene expression (15 days) in the absence of cytotoxic, cytostatic or modulatory effects. Infection of freshly isolated cells was also observed but at lower levels (20%). CONCLUSIONS: Baculovirus vectors can therefore be used to deliver target genes to cultured rat and human HSC with high efficiency and longevity in the absence of detrimental effects on cell function.

Actins↗

PubMed2002

Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors.

Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a 'minigenome' derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but beta-galactosidase activity was limited to 10-20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis delta virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis delta virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host-cell interactions.

Animals↗