Sofosbuvir modulates the intimate relationship between hepatitis C virus and lipids
Authors: Manuel Romero‐Gómez, Ángela Rojas
Journal: Hepatology (2014)

Potential conflict on interest: Dr. Romero‐Gomez consults for and is on the speakers' bureau of Gilead and Roche. See Article on Page 790 The close relationship between hepatitis C virus (HCV) and lipid metabolism blurs the line separating host from viral factors. Lipid genes are expressed depending on the virus, and vice versa, lipids strongly influence life cycle of the virus. In the current issue of Hepatology, Meissner et al. report on the impact of sofosbuvir (SOF) plus ribavirin (RBV) treatment on lipid metabolism and also on expression of genes related to lipid homeostasis in liver of patients with chronic hepatitis C genotype 1. SOF is a nucleotide analog inhibitor of HCV nonstructural (NS)5B polymerase recently approved by the U.S. Food and Drug Administration and European Medicines Agency for treatment of hepatitis C in combination with other antivirals, such as NS5A inhibitors daclatasvir and ledipasvir, or a protease inhibitor such as simeprevir, or even a pegylated interferon (Peg‐IFN) plus RBV‐based triple therapy for treatment of HCV genotypes 1‐6 covering the spectrum of hepatitis C‐related liver diseases. Sustained virological response (SVR) rates are higher than 90% in the majority of scenarios. SOF‐induced HCV‐RNA clearance was associated with early increase in concentration and size of low‐density lipoprotein (LDL) particles, and baseline concentrations predicted response. Furthermore, the investigators demonstrated that the virus modulated the levels of gene expression related to lipid transport, assembly, and metabolism.1 HCV virions circulate in serum as a lipo‐viro‐particle (LVP) that contains HCV‐RNA + structural viral proteins + apolipoprotein (Apo) B‐100 + ApoB‐48 + ApoE + ApoCII + ApoCIII + cholesteryl ester + phospholipids + cholesterol + triglycerides (TGs). Indeed, LVPs can be almost completely precipitated by anti‐ApoB and ‐ApoE antibodies, and removal of lipoproteins by apheresis reduces HCV‐RNA levels by 77%. Indeed, knockdown of ApoE reduces intra‐ and extracellular virus concentrations. This suggests that most of the viral particles are tightly associated with several lipoprotein classes.2 Hepatic lipid content is regulated by the balance between hepatic lipid uptake (function of substrate delivery and transport into the hepatocyte), synthesis (de novo lipogenesis and esterification), oxidation (hepatic oxidative flux), and export (TGs as very‐low‐density lipoprotein [VLDL]).3 Lipids affect several steps of the life cycle, including entry, replication, assembly, and secretion (Fig. 1). Briefly, infectious HCV particles associated with lipoproteins are easily internalized by lipoprotein receptor‐mediated endocytosis. Virus binding and internalization are followed by translation of the HCV‐RNA genome. The HCV core proteins bind to lipid droplets through diacylglycerol O‐acyltransferase 1 (DGAT‐1) and bring together nonstructural (complex replication) and structural proteins. Subsequent to capsid assembly, nascent virions bind into the lumen of the endoplasmic reticulum (ER) and interact in the VLDL secretion pathway. Reduced activity of microsomal triglyceride transfer protein (MTP) results in decreased secretion of VLDL, leading to lipid accumulation.4 Core and NS5A proteins disturb MTP activity in the hepatocyte. MTTP is an essential chaperone for the assembly of VLDL, which transports TGs, phospholipids, and cholesterol in the circulation.Figure 1: Lipids play a key role in each step of the viral life cycle, entry, replication, assembly, and secretion steps. Several proteins have been studied as possible therapeutic targets. (A) Lipid biosynthesis: HCV modulates cellular lipid metabolism to enhance its replication. HCV circulates in the blood in association with lipoproteins. HCV infection is associated with enhanced lipogenesis, reduced secretion, and β‐oxidation of lipids. HCV‐induced imbalance in lipid homeostasis leads to steatosis. Many lipids are crucial for viral life cycle, and inhibitors of cholesterol/fatty acid biosynthetic pathways inhibit viral replication, maturation, and secretion. Farnesyl‐diphosphate farnesyltransferase 1 (FDFT1) is the first specific enzyme in cholesterol biosynthesis, catalyzing the dimerization of two molecules of farnesyl diphosphate in a two‐step reaction to form squalene, essential for lipid‐droplet (LD) formation. Its inhibition by small interfering RNA or YM‐53601 modulated HCV propagation; HCV modulates lipid homeostasis by increasing lipogenesis through SREBP activation and reducing oxidation and lipid export. Inhibiting SREBP activation by treatment with 25‐hydroxycholesterol blocks HCV replication. HMG‐CoA reductase is the rate‐limiting enzyme in the biosynthesis of cholesterol from mevalonic acid. The most effective inhibitors are the statins that improve the SVR rate together with the standard therapy. Acetyl‐coenzyme A acetyltransferase (ACAT): cholesterol ester synthesis, which is destined for storage in LD or for secretion as apolipoprotens essential for LVP formation. ACAT inhibitor TMP‐153 decreases HCV particle production. FASN: Its function is de novo synthesis of fatty acid. FASN is up‐regulated during HCV infection, and the use of C75 (FASN's inhibitor) regulates HCV entry and production. (B) Entry: Cholesterol transporters and lipoprotein receptors play a key role in HCV entry and fusion. Given that viral entry would constitute a key target for antiviral strategies, inhibitor molecules interacting with viral and/or cellular membranes or interfering with the function of lipid metabolism regulators of HCV entry could offer strong antiviral potential. The target of ezetimibe is Niemann‐Pick C1‐like 1 (NPC1L1) protein. Ezetimibe inhibits the absorption of intestinal cholesterol and alpha‐tocopherol. NCPL1L1 protein may play a critical role in regulating lipid metabolism in addition to stopping HCV entry. HCV particles are known to be in complex with lipoproteins. As a result of this interaction, the LDLR has been proposed as a potential entry factor for HCV. CD81 (TAPA‐1), a member of the tetraspanin integral membrane protein family, has been identified as an essential receptor for HCV. CD81 interacts with E2 structural viral protein promoting viral entry. (C) Replication: HCV replication takes place at a unique subcellular compartment, the ER‐derived membranous web, which consists of clusters of HCV‐induced vesicles and LDs and is the proposed site of viral assembly. LD‐binding protein, tail‐interacting protein 47 (TIP47), regulates HCV‐RNA replication through interaction with the viral NS5A protein. Phosphatidylinositol 4‐kinase alpha (PI4KIIIα) forms a "NS5A‐PI4KIIIα complex," which is crucial to form the membranous web site. FBL2 forms a stable immune‐precipitable complex with the HCV protein, NS5A, in a reaction crucial for HCV‐RNA replication. (D) Assembly: Initiation of virion assembly is thought to require release of replicated genomes from such sites to allow contact with core protein, which forms the virion capsid. Because core is located on the cytosolic side of the ER membrane, assembly probably initiates in the cytosol before further maturation, and release occurs by transfer of nascent particles across the ER membrane. Several studies have identified cytosolic storage organelles, termed LDs, and the VLDL assembly pathway that occurs in the ER lumen as major contributors from the host cell to virion assembly. The TG‐synthesizing enzyme, DGAT1, plays a critical role in HCV infection by recruiting the HCV capsid protein core onto the surface of cellular LDs. NS5A trafficking to LDs depends on DGAT1 activity as well. MTP is essential for hepatic secretion of ApoB‐ and E‐containing lipoproteins, in order to complete the VLDL pathway secretion, essential for the HCV. (E) Secretion: Hepatocyte nuclear factor 4 alpha (HNF4‐α), the most abundant transcription factor in the liver, regulates the VLDL secretory pathway. The use of benzafibrate (a chemical inhibitor) suppressed HCV assembly and secretion and may serve as a potential target for the treatment of HCV infection. Abbreviations: CLDN1, claudin‐1; SR‐B1, scavenger receptor class B type 1.The relationship between baseline LDL‐cholesterol (LDL‐C) concentration and the possibility of achieving SVR has been reported largely in patients receiving Peg‐IFN + RBV5 as well as with direct‐acting antiviral‐based triple therapy6 and, in the current article under discussion, with SOF‐based interferon (IFN)‐free regimens.3 Lipid‐conforming LVPs are released after HCV clearance and their concentrations increase in plasma. The higher the baseline LDL‐C level, the greater the chance of curing hepatitis C, a finding that was especially impressive in patients bearing nonfavorable interleukin (IL)28B genotype as well as previous nonresponders to Peg‐IFN + RBV when treated with telaprevir‐based triple therapy.7 IL28B genotype and host serum lipid levels are related in hepatitis C patients, but were shown to be independently associated with sustained response in very‐difficult‐to‐cure patients, suggesting a synergistic effect.8 On the other hand, studies based on transcriptome and proteomic analyses have demonstrated that expression of host genes involved in biosynthesis, lipid peroxidation, and intracellular lipid transport are profoundly altered after HCV infection. Although the most common lipids are TGs, cholesterol, and fatty acids, many other lipid species, such as diacylglycerols, could be involved in metabolic derangements and fibrosis progression. Sterol regulatory element‐binding transcription factor 1 (SREBF1) gene expression levels, which control transcription of genes such as hydroxyl‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase gene (HGMCR) and fatty acid synthase gene (FASN) required for cholesterol biosynthesis, were up‐regulated in liver tissue and in peripheral blood mononuclear cells (PBMCs) and were related to higher levels of intracellular cholesterol.9 Besides, LDL receptor (LDLr) gene expression was found to be down‐regulated, whereas geranylgeranyl pyrophosphate (GPP) levels were up‐regulated, in nonresponder PBMC patients. GPP is necessary for the gernanylgeranylation of cellular protein F‐box and leucine‐rich repeat protein 2 (FBL2), which is necessary in the biosynthetic pathway of cholesterol, which, in turn, is indispensable in forming the HCV replication complex.10 Moreover, the investigators focused on several genes implicated in lipid transport, such as APOB, APOC‐III, APO‐L3, and lipid‐signaling leptin receptor, MTTP together with liver X receptor/retinoid X receptor activation pathways. The changes in these genes not only supported the link between lipids and HCV infection, but also they could identify these genes as therapeutic targets, that is, the inhibition of FASN or DGAT‐1 activity blocked HCV‐RNA replication, production of infectious virus particles, and the entry of the virus into the cell.11 Genes involved in lipid metabolism constitute a long and complex list. Genetic analyses, such as the hypothesis‐free approach, could help, but concordance among studies is weak, and when selecting candidate genes for further research, some important information is lost at each step.13 Furthermore, gene expression in PBMCs may not always match liver expression and, in some cases, confuses the linking of both expressions. SREBF1 gene expression seems to be up‐regulated in the liver, but down‐regulated in PBMCs. An explanation for this phenomenon is, as yet, not available, and we need to be cautious when translating results from PBMCs to liver tissue. An even further gap could appear when translating results from cell culture to PBMCs, or liver. Several studies have observed that the increased expression of IFN‐stimulated genes (ISGs) is associated with treatment failure. Chen et al. suggested that ISG profiling using liver biopsy could be used to predict treatment outcomes before IFN therapy. They noted that gene expressions in responder patients were closer to normal liver expression, whereas, in nonresponders, a general up‐regulation of gene expression was observed.14 However, the pattern of ISG expression in PBMCs is different from that of the liver and may not predict HCV treatment outcomes.15 Interestingly, this pattern remains unchanged in patients receiving a SOF‐based, IFN‐free regimen. ISG expression is dependent on a fine balance of pro‐ and antiviral factors comprising endogenous IFNs, ISG, and cellular and viral microRNAs.16 HCV promotes insulin resistance (IR) by inducing degradation of insulin‐receptor substrate 1 through different pathways, depending on viral genotype.17 IR promotes steatosis and fibrosis progression and also improves viral fitness. Eradication of the virus has been associated largely with an improvement in insulin sensitivity, and decreased risk of impaired fasting glucose, or type 2 diabetes. Meissner et al. monitored glycosylated hemoglobin (HbA1c) and homeostatic model assessment of IR (HOMA‐IR) to test the impact of SOF and RBV on glucose metabolism before, and 24 weeks after, treatment. Their results have been controversial. HOMA‐IR did not change during or after treatment, despite HCV clearance, whereas HbA1c decreased from baseline to end of follow‐up, irrespective of viral response. Explanations for these findings are difficult. Danoprevir, a protease inhibitor, promoted, in monotherapy, a short‐term improvement in insulin sensitivity, in parallel with HCV‐RNA decline in patients with hepatitis C genotype 1.18 A direct effect of this drug on insulin metabolism could not be excluded. Furthermore, any condition that shortens erythrocyte lifespan or decreases mean erythrocyte age (such as RBV) may lower HbA1c and give false‐negative results. No differences in the improvement of glucose metabolism after therapy with SOF + RBV were reported. This aspect could be related to confounder factors, such as the use of drugs able to induce hemolytic anemia and also to the use of HbA1c and HOMA‐IR, as surrogate markers. Nevertheless, all patients achieved viral response on treatment; the impact on glucose homeostasis needs to be addressed in further studies. To improve the usefulness of HOMA‐IR as a surrogate marker of IR requires proper handling of fasting blood samples (which were not guaranteed in the current study), hemolysis of blood sample could result in degradation of insulin, and freezing of samples could result in degradation of C‐peptide and glucose.19 The investigators monitored ApoB/ApoA1 ratio to support a possible role of HCV eradication on cardiovascular disease (CVD) risk. ApoB, as part of the LVP, increased in a similar fashion as LDL‐C, but this change was not sustained at end of treatment and was not different in relation to treatment outcome. The link between HCV infection and CVD risk is intriguing. Confounding variables preclude definite conclusions, and pathogenic mechanisms remain elusive.20 In summary, this elegant study supports the concept of the virus hijacking the machinery responsible for controlling lipid metabolism, the aims being to improve viral fitness, promote disease progression, and avoid final eradication. SOF‐based therapeutic regimens surmount all of these hurdles, eradicate the virus, and restore lipid metabolism. Author names in bold designate shared co‐first authorship.