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At least 217 records · Page 12Linked to original sources
PubMed2006

siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations.

RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

Base Sequence↗
PubMed1994

Molecular analysis of RepHI1A, a minimal replicon of the IncHI1 plasmid R27.

R27, a large conjugative plasmid of the HI incompatibility group, was subjected to a subcloning analysis which revealed the presence of a Poll-independent replicon and determinants contributing to incompatibility within a 2.7 kb SalI/XbaI fragment. The DNA sequence of the minimal replicon revealed the presence of a large open reading frame (ORF) as well two sets of 19 bp repeated oligonucleotides (iterons), in addition to characteristic Escherichia coli origin elements. The protein encoded by the ORF possesses homology with replication initiator proteins encoded by a number of plasmids from different incompatibility groups. Deletion analysis suggested that the iterons are responsible for incompatibility reactions. Dissection of the replicon confirmed this and defined a minimal origin of 230 bp. The putative replication initiator was expressed in an in vitro transcription-translation system, and the 5' end of the mRNA encoding its synthesis was identified. Transcriptional fusion of the repA promoter to lacZ demonstrated an autoregulatory function of RepA. A series of iterons present downstream of the RepA coding sequence are dispensable but are responsible for copy-number control. The minimal replicon appears to be partition-defective.

Amino Acid Sequence↗
PubMed1982

Arrest of chain growth of replicon-sized intermediates by aphidicolin during rat fibroblast cell chromosome replication.

The effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 micrograms/ml) causing 90-95% inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2-5 x 10(-7) Da); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high-molecular-weight DNA. This merging of the intermediates was associated with DNA synthesis in gaps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine-DNA leader with long-wave ultraviolet light; when the cells had been pulse-labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin-arrested intermediates was cut into fragments of the original size by long-wave ultraviolet light irradiation. The arrest of chain elongation at the replicon-size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNAs.

Animals↗
PubMed1997

Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136.

The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis. Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194. The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment. Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids. The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame. Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids. Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136. The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.

Amino Acid Sequence↗
PubMed2006

Distribution of gamma-hexachlorocyclohexane-degrading genes on three replicons in Sphingobium japonicum UT26.

Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 utilizes the important insecticide gamma-hexachlorocyclohexane as a sole source of carbon and energy. In previous studies, we isolated and characterized six structural genes (linA to linF) and one regulatory gene (linR) of UT26 for the degradation of gamma-hexachlorocyclohexane to beta-ketoadipate. Our analysis in this study indicated that the UT26 genome consists of three large circular replicons of 3.6 Mb, 670 kb, and 185 kb. The 3.6 Mb and the 670 kb replicons had one and two copies, respectively, of the 16S ribosomal RNA gene, and these replicons were designated as chromosomes (Chr) I and II, respectively. Chr I was indicated to be a main chromosome carrying the dnaA gene. The first three lin genes, linA to linC, for conversion of gamma-hexachlorocyclohexane to 2,5-dichlorohydroquinone, were dispersed on Chr I. The 185 kb plasmid, pCHQ1, carried the linRED operon for the conversion of 2,5-dichlorohydroquinone to maleylacetate and was conjugatively transferred to another sphingomonad strain. The linF gene encoding maleylacetate reductase was located on Chr II. These results indicated that the genes for the complete gamma-hexachlorocyclohexane degradation are dispersed on the three large replicons of UT26.

Bacterial Proteins↗
PubMed2005

Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons.

Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5'- and 3'-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5'- and 3'-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNA(Val), U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3'/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5'- or 3'-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3'-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNA(Val) as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3'-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNA(Val)-driven Rzs and U6-derived siRNAs both directed against highly conserved 5'- and 3'-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.

Blotting, Western↗
PubMed2006

SCH 503034, a mechanism-based inhibitor of hepatitis C virus NS3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells.

Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may abrogate cellular response to alpha interferon (IFN-alpha) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.

Antiviral Agents↗
PubMed1992

Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association.

Animals↗
PubMed1986

Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing.

New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli. These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells. These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy-number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa. The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 microliter of a lysate containing 10(6) helper phage. The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed. The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs. Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment. Two of these elements also contain the origin-of-transfer sequence oriT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation.

Bacteriophage mu↗
PubMed1987

Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons.

An in vivo cloning system that uses derivatives of the Escherichia coli bacteriophage Mu with plasmid replicons has been extended to five different species of the family Enterobacteriaceae. Mu and these mini-Mu replicon elements were introduced into strains of E. coli, Shigella flexneri, Salmonella typhimurium, Citrobacter freundii, and Proteus mirabilis by infection, by transformation, or by conjugation with newly constructed broad-host-range plasmids containing insertions of these elements. Lysates from these cells, lysogenic for Mu and mini-Mu elements, were used to infect sensitive recipient strains of E. coli, S. typhimurium, and C. freundii. Drug-resistant transductants had mini-Mu replicon elements with inserts of different DNA sequences. All of the lysogens made could be induced to yield high phage titers, including those coming from strains that were resistant to Mu and Mu derivatives. Clones of 10 particular genes were isolated by their ability to complement specific mutations in the recipient strains, even in the presence of the E. coli K-12 restriction system. Some of the mini-Mu replicon elements used contained lac gene fusing segments and resulted in fusions of the lac operon to control regions in the cloned sequences.

Citrobacter↗
PubMed1995

Boundaries of the pSC101 minimal replicon are conditional.

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.

Bacterial Proteins↗
PubMed1997

Suppression of initiation defects of chromosome replication in Bacillus subtilis dnaA and oriC-deleted mutants by integration of a plasmid replicon into the chromosomes.

We constructed Bacillus subtilis strains in which chromosome replication initiates from the minimal replicon of a plasmid isolated from Bacillus natto, independently of oriC. Integration of the replicon in either orientation at the proA locus (115 degrees on the genetic map) suppressed the temperature-sensitive phenotype caused by a mutation in dnaA, a gene required for initiation of replication from oriC. In addition, in a strain with the plasmid replicon integrated into the chromosome, we were able to delete sequences required for oriC function. These strains were viable but had a slower growth rate than the oriC+ strains. Marker frequency analysis revealed that both pyrD and metD, genes close to proA, showed the highest values among the markers (genes) measured, and those of other markers decreased symmetrically with distance from the site of the integration (proA). These results indicated that the integrated plasmid replicon operated as a new and sole origin of chromosome replication in these strains and that the mode of replication was bidirectional. Interestingly, these mutants produced anucleate cells at a high frequency (about 40% in exponential culture), and the distribution of chromosomes in the cells was irregular. A change in the site and mechanism (from oriC to a plasmid system) of initiation appears to have resulted in a drastic alteration in coordination between chromosome replication and chromosome partition or cell division.

Aldehyde Oxidoreductases↗
PubMed1999

Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis.

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.

Bacillus thuringiensis↗
PubMed2005

Novel replicon-based reporter gene assay for detection of rubella virus in clinical specimens.

Proof of concept for a novel diagnostic assay for rubella virus (RUB) based on RUB replicons expressing reporter genes was demonstrated. RUB replicons have the structural protein coding region replaced with a reporter gene such as green fluorescent protein or chloramphenicol acetyltransferase. Previously, it was shown that a replicon construct with a specific in-frame deletion in the nonstructural protein coding region (NotI, approximately nucleotides 1500 to 2100 of the genome) failed to replicate and express the reporter gene unless rescued by a coinfecting wild-type helper RUB (W.-P. Tzeng et al., Virology 289:63-73, 2001). In the present study, it was found that rescue of reporter gene expression by NotI replicons occurred when coinfection was done with clinical specimens containing RUB, indicating that this system could be the basis for a diagnostic assay. The assay was sensitive, using laboratory RUB strains and as low a dose as one plaque-forming unit. The assay was specific in that it was positive for RUB strains of both genotypes and was negative for a panel of human viruses. It was also possible to genetically sequence the RUB present in positive clinical specimens detected in the assay for genotypic strain determination.

Animals↗
PubMed1997

Sindbis virus replicons and Sindbis virus: assembly of chimeras and of particles deficient in virus RNA.

Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy have made them useful as vectors for the expression of heterologous genes. In infected cells, the nonstructural proteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the capsid protein that interacts with the RNA to form the nucleocapsid and the proteins embedded in the lipid envelope, are translated from a subgenomic mRNA and can be replaced by heterologous genes. Such modified genomes are self-replicating (replicons); they can be introduced into the cells by transfection and can also be packaged into extracellular particles with defective helper (DH) RNAs. The particular DH RNA determines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination between the replicon genome and the DH RNA may occur. The studies described here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus genome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second helper to achieve high-level expression of the viral glycoproteins. For this reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross River virus, and the second was to make deletions in that gene to maintain the translational enhancer in the RNA but to eliminate the positively charged region in the protein that should be essential for the specific and nonspecific interactions with RNA. Transfections with replicon RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely devoid of RNA. The inclusion of a helper expressing an intact Sindbis virus capsid protein gene led to the production of high levels of packaged replicons. Recombinants were not detected even after several undiluted passages.

Amino Acid Sequence↗
PubMed2000

Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein.

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55(Gag) elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.

Adenovirus E2 Proteins↗
PubMed2001

Effect of alpha interferon on the hepatitis C virus replicon.

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.

Adaptation, Physiological↗
PubMed2002

Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway.

Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the alpha subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5' noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis.

Activating Transcription Factor 6↗