Hepatitis C virus: The viral way to fatty liver
Authors: Sophie Clément, Francesco Negro
Journal: Journal of Hepatology (2007)

The prevalence of steatosis in chronic hepatitis C patients varies between 40% and 80%, depending on the features of the population studied in terms of alcohol consumption, prevalence of overweight, diabetes and other risk factors of fatty liver [[1]Asselah T. Rubbia-Brandt L. Marcellin P. Negro F. Steatosis in chronic hepatitis C: why does it really matter?.Gut. 2006; 55: 123-130Crossref PubMed Scopus (335) Google Scholar]. If all known factors of fatty liver are excluded, the prevalence of steatosis in chronic hepatitis C is still about 40%. This figure represents an approximately two fold increase compared to the prevalence of steatosis in another common chronic liver disease like hepatitis B (∼20%) [2Czaja A.J. Carpenter H.A. Santrach P.J. Moore S.B. Host- and disease-specific factors affecting steatosis in chronic hepatitis C.J Hepatol. 1998; 29: 198-206Abstract Full Text PDF PubMed Scopus (239) Google Scholar, 3Thomopoulos K.C. Arvaniti V. Tsamantas A.C. Dimitropoulou D. Gogos C.A. Siagris D. et al.Prevalence of liver steatosis in patients with chronic hepatitis B: a study of associated factors and of relationship with fibrosis.Eur J Gastroenterol Hepatol. 2006; 18: 233-237Crossref PubMed Scopus (138) Google Scholar]. Thus, this observation alone suggests that the hepatitis C virus (HCV) may directly cause steatosis, at least in some patients. Steatosis is more frequent and severe in patients with HCV genotype 3 [4Mihm S. Fayyazi A. Hartmann H. Ramadori G. Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype.Hepatology. 1997; 25: 735-739Crossref PubMed Scopus (235) Google Scholar, 5Rubbia-Brandt L. Quadri R. Abid K. Giostra E. Male P.J. Mentha G. et al.Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3.J Hepatol. 2000; 33: 106-115Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 6Adinolfi L.E. Gambardella M. Andreana A. Tripodi M.F. Utili R. Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity.Hepatology. 2001; 33: 1358-1364Crossref PubMed Scopus (964) Google Scholar, 7Castera L. Hezode C. Roudot-Thoraval F. Lonjon I. Zafrani E.S. Pawlotsky J.M. et al.Effect of antiviral treatment on evolution of liver steatosis in patients with chronic hepatitis C: indirect evidence of a role of hepatitis C virus genotype 3 in steatosis.Gut. 2004; 53: 420-424Crossref PubMed Scopus (128) Google Scholar]. This hints at the presence of "steatogenic" sequences across the genome of genotype 3. In other words, this viral genotype, more often and more significantly than other genotypes (albeit not exclusively), may be directly involved in the accumulation of triglycerides in hepatocytes. This interpretation is supported by two additional observations. The first is that, especially in patients with genotype 3, the severity of steatosis correlates with the level of HCV replication, expressed as HCV RNA level in liver [[5]Rubbia-Brandt L. Quadri R. Abid K. Giostra E. Male P.J. Mentha G. et al.Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3.J Hepatol. 2000; 33: 106-115Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar] or in serum [[6]Adinolfi L.E. Gambardella M. Andreana A. Tripodi M.F. Utili R. Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity.Hepatology. 2001; 33: 1358-1364Crossref PubMed Scopus (964) Google Scholar]. Secondly, the fatty liver is significantly reduced or disappears when patients are successfully treated with antivirals, especially in patients with genotype 3, while those with genotypes non-3 may mostly remain steatotic even in the case of sustained virological response [8Kumar D. Farrell G.C. Fung C. George J. Hepatitis C virus genotype 3 is cytopathic to hepatocytes. Genotype-specific reversal of hepatic steatosis after sustained response to antiviral therapy.Hepatology. 2002; 36: 1266-1272Crossref PubMed Scopus (339) Google Scholar, 9Poynard T. Ratziu V. McHutchison J. Manns M. Goodman Z. Zeuzem S. et al.Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C.Hepatology. 2003; 38: 75-85Crossref PubMed Scopus (548) Google Scholar]. A relapse after the end of therapy may result in the reappearance of steatosis in patients in whom it had disappeared while on-treatment [[10]Rubbia-Brandt L. Giostra E. Mentha G. Quadri R. Negro F. Expression of liver steatosis in hepatitis C virus infection and pattern of response to alpha-interferon.J Hepatol. 2001; 35: 307Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. Based on currently available evidence, HCV seems to interfere with lipid metabolism via at least three distinct, non-mutually exclusive mechanisms: impaired secretion, impaired degradation and increased synthesis. Historically, impaired secretion of lipids from the infected hepatocyte was the first proposed mechanism of HCV-induced steatosis. In fact, serum levels of apolipoprotein B (ApoB) and cholesterol are reduced in chronic hepatitis C patients in whom steatosis responds to antiviral therapy [11Serfaty L. Andreani T. Giral P. Carbonell N. Chazouillères O. Poupon R. Hepatitis C virus induced hypobetalipoproteinemia: a possible mechanism for steatosis in chronic hepatitis C.J Hepatol. 2001; 34: 428-434Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 12Hofer H. Bankl H.C. Wrba F. Steindl-Munda P. Peck-Radosavljevic M. Osterreicher C. et al.Hepatocellular fat accumulation and low serum cholesterol in patients infected with HCV-3a.Am J Gastroenterol. 2002; 97: 2880-2885Crossref PubMed Scopus (106) Google Scholar], suggesting that HCV may interfere with the very-low density lipoprotein (VLDL) assembly and/or secretion. The disappearance of fatty liver in sustained virological responders correlates with normalization of cholesterol and ApoB levels [9Poynard T. Ratziu V. McHutchison J. Manns M. Goodman Z. Zeuzem S. et al.Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C.Hepatology. 2003; 38: 75-85Crossref PubMed Scopus (548) Google Scholar, 12Hofer H. Bankl H.C. Wrba F. Steindl-Munda P. Peck-Radosavljevic M. Osterreicher C. et al.Hepatocellular fat accumulation and low serum cholesterol in patients infected with HCV-3a.Am J Gastroenterol. 2002; 97: 2880-2885Crossref PubMed Scopus (106) Google Scholar]. Hypocholesterolemia was reported to be associated with genotype 3 [[12]Hofer H. Bankl H.C. Wrba F. Steindl-Munda P. Peck-Radosavljevic M. Osterreicher C. et al.Hepatocellular fat accumulation and low serum cholesterol in patients infected with HCV-3a.Am J Gastroenterol. 2002; 97: 2880-2885Crossref PubMed Scopus (106) Google Scholar]. Thus, clinical data suggest that HCV interferes with VLDL secretion, a defect corrected by antivirals. In vitro studies and a transgenic mouse model have suggested that the HCV core protein alone is sufficient to induce steatosis [13Barba G. Harper F. Harada T. Kohara M. Goulinet S. Matsuura Y. et al.Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.Proc Natl Acad Sci USA. 1997; 94: 1200-1205Crossref PubMed Scopus (582) Google Scholar, 14Moriya K. Yotsuyanagi H. Shintani Y. Fujie H. Ishibashi K. Matsuura Y. et al.Hepatitis C virus core protein induces hepatic steatosis in transgenic mice.J Gen Virol. 1997; 78: 1527-1531Crossref PubMed Scopus (583) Google Scholar, 15Perlemuter G. Sabile A. Letteron P. Vona G. Topilco A. Chretien Y. et al.Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.FASEB J. 2002; 16: 185-194Crossref PubMed Scopus (521) Google Scholar]. These models have all used genotype 1-derived constructs, but our recent work has reported that type 3a seems more efficient in terms of fat accumulation [[16]Abid K. Pazienza V. De Gottardi A. Rubbia-Brandt L. Conne B. Pugnale P. et al.An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation.J Hepatol. 2005; 42: 744-751Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar], in agreement with clinical observations. In transgenic mice, the HCV core protein inhibits the microsome triglyceride transfer protein (MTP) activity [[15]Perlemuter G. Sabile A. Letteron P. Vona G. Topilco A. Chretien Y. et al.Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.FASEB J. 2002; 16: 185-194Crossref PubMed Scopus (521) Google Scholar]. Since this enzyme plays a key, rate-limiting role in VLDL assembly, the consequence of its inhibition is the accumulation of triglycerides. The intrahepatic levels of MTP mRNA are reduced in chronic hepatitis C patients, especially those with steatosis and/or genotype 3 [[17]Mirandola S. Realdon S. Iqbal J. Gerotto M. Dal Pero F. Bortoletto G. et al.Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis.Gastroenterology. 2006; 130: 1661-1669Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar]. HCV may also cause steatosis by impairing fatty acid oxidation. The expression of the HCV core protein in hepatoma cells causes a down-regulation of the peroxisome proliferator-activated receptor α (PPARα), a nuclear receptor regulating several genes responsible for fatty acid degradation [[18]Yamaguchi A. Tazuma S. Nishioka T. Ohoshi W. Hyogo H. Nomura S. et al.Hepatitis C virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver.Dig Dis Sci. 2005; 50: 1361-1371Crossref PubMed Scopus (97) Google Scholar]. These same authors have also reported a down-regulation of mitochondrial carnitine palmitoyl transferase-1 (CPT-1), the rate-limiting enzyme of mitochondrial β-oxidation, which is the main catabolic pathway of fatty acids, and of the acyl CoA oxidase (AOX). A reduced expression of CPT-1 in the liver of chronic hepatitis C patients has been reported recently [[19]Cheng Y. Dharancy S. Malapel M. Desreumaux P. Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor alpha and carnitine palmitoyl acyl-CoA transferase 1A.World J Gastroenterol. 2005; 11: 7591-7596Crossref PubMed Scopus (62) Google Scholar]. The down-regulation of several genes, such as CPT-1 and AOX, is transcriptionally controlled by PPARα. Thus, the reported effects may be secondary to down-regulation of PPARα. Intrahepatic PPARα mRNA is significantly reduced in the liver of chronic hepatitis C patients [20Dharancy S. Malapel M. Perlemuter G. Roskams T. Cheng Y. Dubuquoy L. et al.Impaired expression of the peroxisome proliferator-activated receptor alpha during hepatitis C virus infection.Gastroenterology. 2005; 128: 334-342Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 21De Gottardi A. Pazienza V. Pugnale P. Bruttin F. Rubbia-Brandt L. Juge-Aubry C.E. et al.Peroxisome proliferator-activated receptor-alpha and -gamma mRNA levels are reduced in chronic hepatitis C with steatosis and genotype 3 infection.Aliment Pharmacol Ther. 2006; 23: 107-114Crossref PubMed Scopus (106) Google Scholar]. Thus, the HCV core protein may modulate the expression of lipid degradation-associated genes via the down-regulation of PPARα. Finally, HCV may induce steatosis via de novo synthesis of fatty acids. HCV upregulates the sterol regulatory element binding protein-1c (SREBP-1c), a transcription factor leading to the up-regulation of enzymes involved in lipid synthesis [[22]Su A.I. Pezacki J.P. Wodicka L. Brideau A.D. Supekova L. Thimme R. et al.Genomic analysis of the host response to hepatitis C virus infection.Proc Natl Acad Sci USA. 2002; 99: 15669-15673Crossref PubMed Scopus (604) Google Scholar]. Chimpanzees experimentally infected with HCV show an increased intrahepatic activity of enzymes involved in lipid synthesis, such as ATP citrate lyase, which are regulated by SREBP-1c [[22]Su A.I. Pezacki J.P. Wodicka L. Brideau A.D. Supekova L. Thimme R. et al.Genomic analysis of the host response to hepatitis C virus infection.Proc Natl Acad Sci USA. 2002; 99: 15669-15673Crossref PubMed Scopus (604) Google Scholar]. The HCV core protein may also bind to and activate the DNA-binding domain of the retinoid receptor α, a transcriptional regulator that controls many cellular functions, including cellular lipid synthesis [[23]Tsutsumi T. Suzuki T. Shimoike T. Suzuki R. Moriya K. Shintani Y. et al.Interaction of hepatitis C virus core protein with retinoid X receptor alpha modulates its transcriptional activity.Hepatology. 2002; 35: 937-946Crossref PubMed Scopus (153) Google Scholar]. In the current issue of the Journal, Jackel-Cram et al. [[24]Jackel-Cram C. Babiuk L.A. Liu Q. Up-regulation of fatty acid synthase promoter by hepatitis C virus core protein: Genotype-3a core has stronger effect than genotype-1b core.J Hepatol. 2007; 46: 999-1008Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar] present their findings on the effect of HCV genotype 1b and 3a core proteins on fatty acid synthase (FAS) promoter and on the molecular mechanisms underlying this process. They have demonstrated that (i) HCV genotype 3a has a stronger effect on FAS promoter activation than core 1b, and that (ii) this activation is primarily mediated by SREBP-1. They have subsequently evaluated the impact on FAS promoter activation of a number of mutations of core proteins, previously described to have important implications either on the processing of core or on its localization around lipid droplets. While it is well documented that regulated processing of core plays crucial roles in determining many of its functions [25Bergqvist A. Rice C.M. Transcriptional activation of the interleukin-2 promoter by hepatitis C virus core protein.J Virol. 2001; 75: 772-781Crossref PubMed Scopus (69) Google Scholar, 26Moorman J.P. Prayther D. McVay D. Hahn Y.S. Hahn C.S. The C-terminal region of hepatitis C core protein is required for Fas-ligand independent apoptosis in Jurkat cells by facilitating Fas oligomerization.Virology. 2003; 312: 320-329Crossref PubMed Scopus (36) Google Scholar, 27Yamanaka T. Kodama T. Doi T. Subcellular localization of HCV core protein regulates its ability for p53 activation and p21 suppression.Biochem Biophys Res Commun. 2002; 294: 528-534Crossref PubMed Scopus (53) Google Scholar, 28Yamanaka T. Uchida M. Doi T. Innate form of HCV core protein plays an important role in the localization and the function of HCV core protein.Biochem Biophys Res Commun. 2002; 294: 521-527Crossref PubMed Scopus (32) Google Scholar], the results reported in this study indicated that this feature of core was differentially involved in FAS promoter activation. For genotype 3a, partial alteration of core protein processing resulted in significantly higher FAS promoter activity, whereas fully processed core was no longer able to up-regulate FAS promoter. For genotype 1b, partial alteration of core protein processing had no effect on FAS, but truncated core protein highly increased FAS promoter activity. In addition, localization of core at lipid droplets did not appear to play a major role. Indeed, the mutation of proline knot or deletion of Y/FATG sequences, both motifs known to be crucial for lipid droplet localization [29Hope R.G. McLauchlan J. Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein.J Gen Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar, 30Hope R.G. Murphy D.J. McLauchlan J. The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins.J Biol Chem. 2002; 277: 4261-4270Crossref PubMed Scopus (143) Google Scholar], had the opposite effect on FAS promoter activation. On the other hand, the last observation that phenylalanine 164 residue within the FATG lipid droplet localization sequence of genotype 3a only appeared to play a major role in FAS activation may have important implications for the understanding of the higher prevalence and severity of steatosis in HCV genotype 3a infection. It has to be said that the search for the viral sequences responsible for the genotype-specific effects on triglyceride accumulation is far from being concluded. The core protein can be divided in three distinct regions, according to the hydrophobic profile predicted by the amino acid sequence. The first region (residues 1–120) is hydrophilic, with clusters of basic residues (lysine and arginine). The domain 2 (residues 121–174, i.e. the C-terminus of the mature core protein) is more hydrophobic, and even more so is the third domain (residues 175–191), cleaved by host proteases [[31]McLauchlan J. Lemberg M.K. Hope G. Martoglio B. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets.EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (397) Google Scholar]. Mutagenesis experiments have shown that domain 2 is responsible for the association with lipids via hydrophobic interactions [[29]Hope R.G. McLauchlan J. Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein.J Gen Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar]. Sequence similarities have been found between this domain and a region involved in lipid binding of oleosin, a protein associated with lipid bodies in plants: the two prolines at positions 138 and 143 and a YATG motif are required for lipid association [[30]Hope R.G. Murphy D.J. McLauchlan J. The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins.J Biol Chem. 2002; 277: 4261-4270Crossref PubMed Scopus (143) Google Scholar]. When we mutated the phenylalanine 164 into a tyrosine in our in vitro expression system [[16]Abid K. Pazienza V. De Gottardi A. Rubbia-Brandt L. Conne B. Pugnale P. et al.An in vitro model of hepatitis C virus genotype 3a-associated triglycerides accumulation.J Hepatol. 2005; 42: 744-751Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar], we failed to observe a significant shift in the ability of genotype 3a to induce triglyceride accumulation. In addition, our 3a-transfected Huh-7 cells were not capable of accumulating triglycerides in a lipid-deprived medium, indirectly ruling out virally induced increase of fatty acid neosynthesis. The discrepant results with those of Jackel-Cram et al. [[24]Jackel-Cram C. Babiuk L.A. Liu Q. Up-regulation of fatty acid synthase promoter by hepatitis C virus core protein: Genotype-3a core has stronger effect than genotype-1b core.J Hepatol. 2007; 46: 999-1008Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar] may be reconducted to subtle metabolic differences among different Huh-7 cell clones. On the other hand, recent data reported by Jhaveri et al. [[32]Jhaveri R. McHutchison J.G. Patel K. Diehl A.M. The role of hepatitis C genotype 3 core protein domain 3 in intrahepatic steatosis.Hepatology. 2006; 44: 300AGoogle Scholar] suggest that specific combinations of mutations (in the domain 3 of the core sequence) may be associated with the steatotic phenotype. Further work is warranted, using models based on the expression of the full-length HCV polyprotein. In addition, use of chimeric, intergenotypic constructs [[33]Pietschmann T. Lohmann V. Kaul A. Krieger N. Rinck G. Rutter G. et al.Persistent and transient replication of full-length hepatitis C virus genomes in cell culture.J Virol. 2002; 76: 4008-4021Crossref PubMed Scopus (308) Google Scholar] may help elucidating the role of other viral proteins in facilitating the fat accumulation. The authors' experimental work cited in the present manuscript is supported by Grant No. 3200B0-103727 from the Swiss National Science Foundation.