Hepatitis C Virus Protein Expression Induces Apoptosis in HepG2 Cells

Kalkeri, Gururaj; Khalap, Nutan; Garry, Robert F.; Fermin, César D.; Dash, Srikanta

doi:10.1006/viro.2000.0835

Cited by 34 publications

(21 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Additionally, some cells exhibited discrete hyperplastic modifications of ER cisternae or altered mitochondria of abnormal, irregular shape which were sometimes vacuolized or with thinned and fragmented cristae similar to those observed by others (not shown) (2). Particular morphological alterations of nuclei potentially caused by apoptosis as described by others for HepG2 cell transfected with full-length HCV RNA (37) could not be detected.…”

Section: Establishment Of Huh-7 Cells Carrying Sfl Genomessupporting

confidence: 77%

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Pietschmann

Lohmann

Kaul

et al. 2002

J Virol

330

257

View full text Add to dashboard Cite

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these fulllength HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.The hepatitis C virus (HCV) was identified as the causative agent for most posttransfusion and sporadic non-A, non-B hepatitis cases (11,45). According to recent estimates, about 170 million individuals worldwide are infected. One striking characteristic of HCV is its strong propensity to persist in the infected host, which often leads to severe liver damage, ranging from chronic hepatitis to liver cirrhosis and even hepatocellular carcinoma (33). The possible immune evasion strategies that allow persistent viral replication in the presence of the host's immune response are not well understood, but the high variability of the virus appears to be a key determinant (38). As a consequence, HCV isolates exhibit marked sequence diversity and have been grouped according to phylogenetic analysis into six different genotypes which together form the genus Hepacivirus within the family Flaviviridae (60).HCV particles are enveloped, have a diameter of 55 to 65 nm, and harbor an ϳ9,600-nucleotide-long plus-strand RNA genome. It carries a s...

show abstract

Section: Establishment Of Huh-7 Cells Carrying Sfl Genomessupporting

confidence: 77%

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Pietschmann

Lohmann

Kaul

et al. 2002

J Virol

330

257

View full text Add to dashboard Cite

show abstract

“…Overexpression of HCV glycoproteins activates grp78 and grp94 promoters in transient gene expression assays, consistent with the induction of an ER stress response (24). In addition, expression of HCV proteins in a hepatoma cell line (HepG2) induces apoptosis (21). Therefore, it is tempting to speculate that BVDV-induced ER stress is mediated through signals that emanate from the ER lumen and are caused by the presence of unfolded or unassembled viral glycoproteins.…”

Section: Discussionmentioning

confidence: 99%

Replication of a Cytopathic Strain of Bovine Viral Diarrhea Virus Activates PERK and Induces Endoplasmic Reticulum Stress-Mediated Apoptosis of MDBK Cells

Jordan

Wang

Graczyk

et al. 2002

J Virol

164

138

View full text Add to dashboard Cite

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca 2؉homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2␣, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

show abstract

“…Although it has been shown that core protein modulates the death receptor pathway of apoptosis, the activation of other apoptosis pathways by HCV proteins has not been analyzed. Interestingly, it has been reported that expression of the complete structural region or the full-length genome may directly induce cytotoxicity in osteosarcoma [25] and hepatoma cell lines [17].…”

mentioning

confidence: 99%

Expression of HCV E1 Protein in Baculovirus-Infected Cells: Effects on Cell Viability and Apoptosis Induction

Ciccaglione

Marcantonio²,

Costantino³

et al. 2003

Intervirology

View full text Add to dashboard Cite

The molecular mechanisms of pathogenesis in hepatitis C virus (HCV) infection are not yet understood. Recently, we reported that the expression of the envelope protein E1 is toxic for Escherichia coli cells. The toxicity is related to the ability of C-terminal transmembrane (TM) domain of E1 to modify membrane permeability. In this study we expressed the E1 protein, complete (a.a. 192–383) or deleted (a.a. 192–340) of the TM region, fused to the C-terminus of glutathione-S-transferase by two recombinant baculoviruses. Infection of Sf9 insect cells by E1 baculovirus induced a rapid decrease in cell viability in the first 18–24 h postinfection. Premature cytopathic changes and low level of E1 protein expression were also reported. The analysis of DNA isolated from cells revealed a typical internucleosomal ladder pattern characteristic of apoptosis. The DNA degradation was first detected at 18 h postinfection by ethidium bromide gel electrophoresis and was confirmed by TUNEL assay. The results indicated that the C-terminal domain of E1 is essential for apoptosis induction as neither cell death nor DNA degradation were observed following infection with the recombinant baculovirus expressing the C-terminal-deleted E1. These findings support the hypothesis that the TM domain of E1 may play a role in viral pathogenesis.

show abstract

Hepatitis C Virus Protein Expression Induces Apoptosis in HepG2 Cells

Cited by 34 publications

References 29 publications

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Replication of a Cytopathic Strain of Bovine Viral Diarrhea Virus Activates PERK and Induces Endoplasmic Reticulum Stress-Mediated Apoptosis of MDBK Cells

Expression of HCV E1 Protein in Baculovirus-Infected Cells: Effects on Cell Viability and Apoptosis Induction

Contact Info

Product

Resources

About