Activation of the CKI-CDK-Rb-E2F Pathway in Full Genome Hepatitis C Virus-expressing Cells

Abstract

Hepatitis C virus (HCV) causes persistent infection inhepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused upregulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.Hepatitis C virus (HCV) 1 causes the persistent infection chronic hepatitis in most infected patients. This disorder eventually progresses to cirrhosis and hepatocellular carcinoma (HCC). Numerous studies have provided evidence supporting a link between chronic HCV infection and HCC (1, 2). However, exactly how HCV infection could be directly involved in the development of HCC remains unclear because of the lack of an efficient in vitro infection system.HCV is a member of the Flaviviridae family and has a positive-strand RNA genome (ϳ9.6 kb). Viral proteins are synthesized as a single polyprotein, which is then cleaved into structural (core, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. In vitro expression of HCV core protein reportedly influences cell growth regulation, and the protein can interact with the cytoplasmic tail of a lymphotoxin-␤ receptor (3), the death domain of tumor necrosis factor receptor-1 (4), and NF-B (5), resulting in enhancement or inhibition of Fas-and tumor necrosis factor-␣-mediated apoptosis. HCV structural protein inhibits Fas-mediated apoptosis by inhibiting cytochrome c release from mitochondria in mice (6). Cellular transformation has been shown to be caused by HCV core protein in the presence of the ras gene (7,8) and by loss of function of LZIP (9) and the presence of STAT3 (signal transducer and activator of transcription-3) (10). NS3 (11) and NS4B (12) proteins reportedly display tumorigenicity in the presence of ras. These results indicate that expression of individual HCV proteins does not cause cellular transformation in vitro. HCV core protein transgenic mice reportedly show induced steatosis and, after 16 months of age, develop HCC (13). In contrast, HCV structural protein transgenic mice do not display neoplastic or cancerous lesions in the liver by 20 months of age (14). Moreover, conditional expression of an HCV structural protein region (nucleotides 294 -3435) causes hepatic injury in transgenic mice (15), but HCC is not observed by at least 16 months of age (data not shown). The frequency of HCC occurrence is reportedly higher in full-...

Down-regulation of the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA polymerase

“…and S.M.L., unpublished data), indicating that Rb is negatively regulated by different strains of HCV that are widely divergent genetically. These findings differ significantly from those reported by TsukiyamaKohara et al (28), who described an initially positive then negative effect on Rb after expression of HCV proteins in HepG2 cells. We found that the negative regulation of Rb abundance by replicating HCV RNA is due to the NS5B RNA-dependent RNA polymerase.…”

Hepatitis C Virus Causes Uncoupling of Mitotic Checkpoint and Chromosomal Polyploidy through the Rb Pathway

“…The overexpression of Mad2, while Mad1 is unaffected, will disturb the normal ratio of these components in the mitotic checkpoint complex (14). The detailed mechanism of Rb suppression by core protein remains obscure (4,13,61). Our studies showed that the core protein inhibits Rb transcription through the suppression of the Rb promoter activity and that core protein is the only HCV protein capable of inhibiting Rb transcription.…”

“…Furthermore, core protein has been shown to impair cell cycle regulation in stably transformed Chinese hamster ovary cells (16). Core protein also affects the function of human Rb, LZIP (a homologue to the Drosophila melanogaster BBF2/dCREB-A protein), and other cell growth regulatory proteins, such as 14-3-3 (1,4,13,20,61). The Rb gene can uncouple cell cycle progression from mitotic control by activation of mitotic checkpoint protein Mad2, leading to genomic instability (14).…”

Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner

“…Briefly, following 30 min of preincubation without ATP, CTP, or UTP, 100 nM HCV RdRp was incubated in 50 mM Tris-HCl (pH 8.0), 200 mM monopotassium glutamate, 3.5 mM MnCl 2 , 1 mM dithiothreitol (DTT), 0.5 mM GTP, 50 M ATP, 50 M CTP, 5 M [␣-32 P]UTP, 200 nM RNA template (SL12-1S), 100 U/ml human placental RNase inhibitor, and the lipid (amount indicated below) at 29°C for 90 min. 32 P-labeled RNA products were subjected to 6% polyacrylamide gel electrophoresis (PAGE) containing 8 M urea. The resulting autoradiograph was analyzed with a Typhoon Trio plus image analyzer (GE).…”