Hepatitis C Virus Selectively Perturbs the Distal Cholesterol Synthesis Pathway in a Genotype-Specific Manner Paul J. Clark,1,2 Alexander J. Thompson,1 David M. Vock,1 Lisa E. Kratz,3 Adviye A. Tolun,4 Andrew J. Muir,1 John G. McHutchison,1 Mani Subramanian,5 David M. Millington,4 Richard I. Kelley,3 and Keyur Patel1 Hepatitis C virus (HCV) subverts host cholesterol metabolism for key processes in its lifecycle. How this interference results in the frequently observed, genotype-dependent clinical sequelae of hypocholesterolemia, hepatic steatosis, and insulin resistance (IR) remains incompletely understood. Hypocholesterolemia typically resolves after sustained viral response (SVR), implicating viral interference in host lipid metabolism. Using a targeted cholesterol metabolomic platform we evaluated paired HCV genotype 2 (G2) and G3 patient sera for changes in in vivo HCV sterol pathway metabolites. We compared HCV genotypic differences in baseline metabolites and following antiviral treatment to assess whether sterol perturbation resolved after HCV eradication. We linked these metabolites to IR and urine oxidative stress markers. In paired sera from HCV G2 (n 5 13) and G3 (n 5 20) patients, baseline sterol levels were lower in G3 than G2 for distal metabolites (7-dehyrocholesterol (7DHC) 0.017 versus 0.023 mg/dL; Padj 5 0.0524, cholesterol 140.9 versus 178.7 mg/dL; Padj 5 0.0242) but not the proximal metabolite lanosterol. In HCV G3, SVR resulted in increased levels of distal metabolites (cholesterol [D55.2 mg/dL; Padj 5 0.0015], 7DHC [D0.0075 mg/dL; Padj 5 0.0026], lathosterol [D0.0430 mg/dL Padj 5 0.0405]). In contrast, lanosterol was unchanged after SVR (P 5 0.9515). Conclusion: HCV G3, but not G2, selectively interferes with the late cholesterol synthesis pathway, evidenced by lower distal sterol metabolites and preserved lanosterol levels. This distal interference resolves with SVR. Normal lanosterol levels provide a signal for the continued proteolysis of 3-hydroxyl-3-methylglutaryl coenzyme A reductase, which may undermine other host responses to increase cholesterol synthesis. These data may provide a hypothesis to explain why hypocholesterolemia persists in chronic HCV infection, particularly in HCV G3, and is not overcome by host cholesterol compensatory mechanisms. (HEPATOLOGY 2012;56:49-56)

T

he hepatitis C virus (HCV) interferes with host lipid metabolism for key viral processes such as cellular entry, replication, and virion release, frequently resulting in relative hypolipidemia, increased hepatic steatosis, and insulin resistance (IR).1-3 These met-

abolic sequelae are important, as they are associated with more advanced liver fibrosis progression and poorer HCV treatment responses.4-8 Viral eradication results in restoration of serum cholesterol and apolipoprotein levels, further implicating a possible direct role for the virus.9

Abbreviations: ALT, alanine aminotransaminase; 7-DHC, 7-dehyrocholesterol; ER, endoplasmic reticulum; G2/G3, genotype 2/3; GC/MS, gas chromatography/ mass spectrometry; HCV, hepatitis C virus; HMGCoA, 3-hydroxyl-3-methylglutaryl coenzyme A; SCAP, SREBP cleavage activating protein; SRB1, scavenger receptor class B type I; SREBP, sterol regulatory element binding protein. From the 1Duke Clinical Research Institute and Department of Gastroenterology, Duke University Medical Center, Durham, NC, USA; 2Kirby Institute (formerly the National Centre in HIV Epidemiology and Clinical Research), University of New South Wales, Sydney, Australia; 3Clinical Mass Spectrometry Laboratory, Kennedy Krieger Institute, Johns Hopkins University, Baltimore, MD, USA; 4Biochemical Genetics Laboratory, Pediatrics, Medical Genetics Division, Duke University Medical Center, Durham, NC, USA; 5Human Genome Sciences, Rockville, MD, USA. Received October 12, 2011; accepted January 23, 2012. Funded by the Duke Clinical Research Institute. Drs. Clark and Thompson received funding from the Duke Clinical Research Institute, the Richard Boebel Family Fund, the National Health and Medical Research Council of Australia (P.J.C.: APP1017139; A.J.T.: APP567057) and the Gastroenterological Society of Australia. Dr. Thompson received funding from the Royal Australian College of Physicians. Dr. Clark received funding from American Association for the Study of Liver Diseases/LIFER Clinical and Translational Research Fellowship in Liver Diseases Award and the National Centre in HIV Epidemiology and Clinical Research (now the Kirby Institute for Infection and Immunity in Society), University of New South Wales, Australia. 49

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The predominance of metabolic effects such as hypocholesteremia and hepatic steatosis with HCV genotype 3 (G3) infection,10 and IR and diabetes with non-G3 genotypes such as HCV G211 and genotypes 1 and 4,12 suggests viral genotype-specific differences in the nature of this interference.13 Direct viral perturbation of cholesterol biosynthetic pathways and their regulation is suggested from experimental in vitro and animal models.14-18 HCV-mediated oxidative stress has been suggested as a possible causative mechanism leading to both HCV-associated hepatic steatosis and IR, and has also been linked to cholesterol regulation.15,19 Finally, from the host perspective, it is not known why compensatory regulation to increase cholesterol synthesis appears inadequate to resolve HCV-associated hypolipidemia. Metabolomics uses methods such as gas chromatography/mass spectrometry (GC/MS) to measure individual metabolite levels and their relative contribution to biosynthetic pathways.20 Metabolomics has been successfully used to identify hereditary sterol defects, where enzyme deficiencies perturb key processes causing elevated levels of preceding sterols and decreased downstream cholesterol synthesis.21,22 All cholesterol synthesis must pass through the final steps of the isoprenoid metabolic pathway, which are exclusively dedicated to cholesterol synthesis (Fig. 1). These steps occur after cyclization of squalene, with subsequent major metabolites: (from proximal to distal in the pathway) being lanosterol, lathosterol, 7-dehyrocholesterol (7-DHC), and cholesterol. Cholesterol cannot be synthesized except by passing through these metabolic steps, and these metabolites are exclusively dedicated to cholesterol. One mechanism of regulatory feedback on cholesterol synthesis occurs by way of direct sensing of the levels of sterol metabolites lanosterol (regulating cholesterol synthesis by way of proteolysis of HMG CoA reductase [3-hydroxy-3-methyl-glutaryl CoA reductase, HMGCoAR] and cholesterol [by way of sterol regulatory element binding proteins, SREBPs]).23-26 Thus, metabolomics has the potential to identify key points in the cholesterol synthesis pathway that might be targeted by HCV, and also to stimulate hypotheses as to how this perturbation might impact cholesterol regulation mechanisms. Metabolomics has not previ-

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ously been used to characterize cholesterol synthesis in HCV-infected patients, or in paired analysis of patient samples before and after successful treatment to estimate the direct viral effect on sterol pathways in the same patient. Our aims were to (1) identify key sites of HCV interference in the sterol pathway to improve our understanding of the interaction between HCV and cholesterol metabolism; (2) assess HCV genotypic differences in baseline sterol metabolism between HCV G3 and non-G3; (3) evaluate changes in sterol metabolites after eradication of virus; and (4) assess sterol metabolites in relation to oxidative stress metabolites and IR.

Patients and Methods Patient Selection. Forty-three treatment-naı¨ve patients chronically infected with HCV-G2 and HCV-G3 were enrolled in a randomized, open label, phase 2 trial of albinterferon alpha-2b and ribavirin (NCT0056006, Human Genome Sciences).27 In accordance with the study protocol, fasting blood and urine samples were collected prospectively at baseline and 12 weeks posttreatment follow-up. HCV RNA levels were measured using reverse-transcription polymerase chain reaction (RT-PCR) (COBAS Ampliprep/COBAS Taqman HCV RNA Assay, Roche Diagnostics, Basel, Switzerland), with a dynamic range of 43-69 million IU/mL. Fasting insulin and glucose levels were tested and IR defined by the Homeostasis Model of Assessment – Insulin Resistance (HOMA-IR, calculated as fasting insulin (lU/mL)  fasting glucose (mmol/L)/22.5).28 IR was assessed at a HOMA-IR threshold >2. Thirty-six patients had paired pre- and posttreatment plasma and urine available for metabolomic analysis. Three patients on HMGCoA reductase inhibitors (or other lipid-lowering therapy) were excluded, leaving 33 patients for analysis. In keeping with clinical practice, liver biopsy was not undertaken per protocol in these G2 and G3 patients, thus precluding analysis of metabolomic relationships to hepatic steatosis. All patients provided written informed consent and this study had local Institutional Review Board approval. Gas Chromatography/Mass Spectrometry. Metabolites in the postsqualene cholesterol synthetic pathway

Address reprint requests to: Keyur Patel, MD, Duke Clinical Research Institute and Duke University Medical Center, PO Box 17969, Durham, NC 27715. E-mail: [email protected]; fax: 919-668-7164. C 2012 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.25631 Potential conflict of interest: Dr. Muir consults for and received grants from Merck, Vertex, Bristol-Myers Squibb, GlaxoSmithKline, and Scynexis. He consults for Achillion and received grants from Gilead, Pfizer, and Abbott. Additional Supporting Information may be found in the online version of this article.

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uninfected (posttreatment) samples for the same individuals. This allowed us to assess the impact of HCV infection on each individual’s cholesterol pathway by using the posttreatment SVR sample as an uninfected ‘‘control’’ for comparison. We conducted the same analysis in patients who failed to achieve SVR to exclude a treatment effect. Individual metabolite levels and proportional contribution to total sterols were evaluated. Baseline sterol metabolites and oxidative stress metabolites were compared between HCV genotypes using the Mann Whitney U Test. Differences between pre- and posttreatment were compared using the Wilcoxon Rank Sum test for paired data, enabling comparison of metabolites for each individual pre-/ posttreatment. To qualify significant results and minimize false-positive associations, correction for multiple testing was conducted using the Hommel method.29 However, given the small sample size and hypothesisgenerating nature of this analysis, both adjusted and unadjusted P values are presented and discussed. Statistical significance was defined as a <0.05. All statistical analysis was performed with SAS 9.2 and Enterprise Guide 4.3 (Cary, NC). Fig. 1. Cholesterol synthesis pathway. Shown here are a restricted number of key metabolites in the cholesterol synthesis pathway. Proximal metabolites contribute to other sterol and nonsterol isoprenoids; however, all cholesterol must pass through the final metabolic steps: (proximal to distal) lanosterol, lathosterol, 7-dehydrocholesterol, and, finally, cholesterol. The sterol metabolites lanosterol and cholesterol are directly sensed by membrane-bound proteins in the endoplasmic reticulum, providing feedback to key regulators of cholesterol synthesis HMGCoA reductase and SREBP, respectively. HMGCoAR, 3-hydroxy-3methyl-glutaryl CoA reductase; SREBP, sterol regulatory element binding protein.

were quantified using ion-ratio GC/MS on an Agilent 6390N/5973 GC/MS system.20 Key sterol metabolites lanosterol, lathosterol, 7-DHC, and cholesterol were assessed from blood samples in the primary analysis. Secondary analysis of other intermediate metabolites was undertaken to attempt to better define specific points of perturbation by HCV (Supporting Fig. 1). After correction for creatinine clearance, baseline oxidative stress metabolites allantoin, 2,3 dinor isoprostane-F2, and isoprostane-F2-alpha were assessed from urine samples using ultra performance liquid chromatography and tandem mass spectrometry. Statistical Analysis. Baseline, pretreatment sterol metabolites were compared between HCV G2 and G3. For each patient, paired pre- and posttreatment serum metabolite levels were then also compared. Thus, for patients who achieved sustained viral response (SVR, HCV RNA negative 12 weeks after treatment completion, n ¼ 24), we were able to compare sterol metabolites in HCV infected (pretreatment) serum with

Results Patient Demographics. There were 33 CHC patients (HCV G2 ¼ 13, HCV G3 ¼ 20) included in this study. Baseline patient characteristics are summarized in Table 1. No patient had a history of diabetes mellitus. Four patients identified as Asian, one as Native American, and the remainder of patients were Caucasian. HCV G2 patients had significantly higher mean HOMA-IR than G3 (3.07 versus 1.96; P ¼ 0.03); however, treatment response did not differ according to pretreatment HOMA-IR (mean HOMAIR, 2.82 versus 3.83; P ¼ 0.0982, in patients achieving SVR versus non-SVR, respectively). Baseline Distal Pathway Sterol Metabolites Are Lower in Patients With HCV G3 Infection But There Are No HCV Genotype-Based Differences in Proximal Sterol Metabolites. Before treatment, HCV G3 was associated with lower median total cholesterol levels than HCV G2 (140.9 versus 178.7 mg/dL; Puncorr ¼ 0.0061, Pcorr ¼ 0.0242, respectively; Table 2). A greater proportion of patients with HCV G3 infection had hypocholesterolemia relative to HCV G2 (<140 mg/dL, 55% versus 15%, respectively, P ¼ 0.0325). There was a marginal difference between genotypes for cholesterol’s precursor metabolite 7-DHC that did not reach the statistical threshold for significance after correction for multiple testing (0.017 versus 0.023

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Table 1. Demographic and Clinical Characteristics of Cohort N BMI (kg/m2) mean (þ/- SD) HOMA-IR median (25th-75th centile range) ALT (U/L) Median (25th-75th centile range) HCV RNA (log10IU/mL) median (25th-75th centile range)) Baseline serum cholesterol (mg/dL) median (25th-75th centile range) Baseline serum triglycerides (mg/dL) median (25th-75th centile range) SVR N (%)

All Patients

HCV-G2

HCV- G3

P

33 28.0 (þ/ 5.6) 2.18 (1.86) 84 (64) 6.18 (1.22) 156 (54) 87 (66) 24 (73)

13 29.6 (þ/ 6.2) 3.07 (4.6) 65 (70) 6.11 (0.8) 187 (24) 138 (47) 10 (77)

20 26.9 (þ/ 5.0) 1.96 (2.0) 84 (59) 6.32 (1.30) 138 (56) 75 (51) 14 (70)

0.1894* 0.0326† 0.4281† 0.9855† 0.0060† 0.0677† 0.9710‡

N, number of patients; HCV, Hepatitis C virus; G2/G3, genotype 2/3; SD, standard deviation, BMI, body mass index; HOMA-IR, Homeostasis Model of Assessment - Insulin Resistance; ALT, alanine aminotransferase; SVR, sustained viral response. *Student t test. †Wilcoxon Rank Sum. ‡Pearson chi-square.

mg/dL; Puncorr ¼ 0.0175, Pcorr ¼ 0.0524 for HCV G3 and G2, respectively). Lower baseline cholesterol correlated with higher baseline HCV viral RNA for G3 (Spearman correlation R ¼ 0.55789; P ¼ 0.0106) but not in patients with HCV G2 infection. Despite the lower baseline cholesterol levels (6possibly lower 7-DHC levels), there were clearly no genotype-specific differences in proximal pathway sterol metabolites (lathosterol or lanosterol, Puncorr ¼ 0.2328 and Puncorr ¼ 0.2297, respectively) suggesting HCV interference may occur distal to these steps in the pathway. We conducted subanalysis of a number of metabolites between lanosterol and lathosterol and the ‘‘accessory pathway’’ to cholesterol by way of desmosterol and found no differences between HCV genotypes. Given the possibility that a preexisting confounding difference in patients could explain the difference seen between HCV genotypes, we compared sterol metabolites in patients after SVR (uninfected sera), differentiating patients by the baseline genotype. There were no serum metabolite differences between these groups in uninfected patients (P ¼ 0.8836), suggesting an unidentified host confounder is less likely to influence the genotype differences seen in baseline sterol metabolites. We hypothesized that if these observed differences in distal pathway sterol metabolites (cholesterol 6 7DHC) were due to HCV, then eradication of the virus (SVR) should result in increases in these distal metabolite levels.

Viral Eradication Results in Significant Increases in Distal Pathway Sterol Metabolites But No Change in the Level of the Proximal Sterol Lanosterol. Viral eradication resulted in significant increases in median cholesterol, 7-DHC, and lathosterol levels for HCV G3 (Table 3). With the release of any postulated HCV-induced, distal block, an increase in total sterol synthesis may expect to increase all metabolites in the pathway. Thus, interestingly, absolute lanosterol level was clearly unchanged after SVR (Pcorr/uncorr ¼ 0.9515), and lanosterol’s contribution to total sterol synthesis may have possibly decreased somewhat for HCV G3 (Pcorr ¼ 0.1014, Puncorr ¼ 0.0419) (Supporting Table 1). To account for an interferon (IFN) treatment effect we also compared metabolites pre- versus posttreatment in patients who did not achieve SVR. In contrast to patients with SVR, metabolite levels were not significantly changed at follow-up after non-SVR. The increase in distal metabolites levels without a change in lanosterol’s level after viral eradication further implicates a HCV-mediated downstream block (i.e., postlanosterol) of cholesterol synthesis. Resolution of these changes occurs with HCV eradication, but not in those patients with persistent HCV infection. Oxidative Stress Metabolites Correlate with Sterol Metabolites and Alanine Aminotransaminase (ALT) Levels. Oxidative stress is a putative mechanism implicated in the viral disruption of host metabolism. Levels

Table 2. Hepatitis C Virus Genotype 2/3-Based Differences in Baseline Sterol Metabolites HCV G2

Cholesterol (mg/dL) median (mean, SD) 7-Dehydrocholesterol (mg/dL) median (mean, SD) Lathosterol (mg/dL) median (mean, SD) Lanosterol (mg/dL) median (mean, SD)

178.7 0.023 0.164 0.009

(183.9, (0.027, (0.210, (0.010,

*After correction for multiple testing using the Hommel method. HCV, Hepatitis C virus; G2/G3, genotype 2/3; SD, standard deviation.

HCV G3

31.8) 0.014) 0.157) 0.004)

140.9 0.017 0.127 0.007

(145.0, (0.017, (0.140, (0.008,

34.0) 0.007) 0.069) 0.004)

P

Adjusted P*

0.0061 0.0175 0.2383 0.2297

0.0242 0.0524 0.2383 0.2383

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Table 3. Comparison of Changes in Metabolite Levels Pre- vs. Posttreatment in Patients With Sustained Viral Response (SVR), SVR by HCV Genotype, and Non-SVR, and Comparing Change in Patients With SVR vs. Non-SVR Pre- vs. Posttreatment Median Metabolite Levels All SVR (n ¼ 24)

Cholesterol 7-Dehydro-Cholesterol Lathosterol Lanosterol

SVR HCV G2 (n ¼ 10)

SVR HCV G3 (n ¼ 14)

SVR vs. Non-SVR Non-SVR (n ¼ 9)

(mg/dL)

P†

(mg/dL)

P†

(mg/dL)

P†

(mg/dL)

P†

All patients (n ¼ 33) P†

35.5 0.005 0.020 0.0001

0.0001* 0.0026* 0.0157* 0.5045

17.6 0.0002 0.014 0.0014

0.1953 0.4922 0.4922 0.4922

55.2 0.0075 0.043 0.0016

0.0015* 0.0026* 0.0405* 0.9515

55.7  0.0258 0.395 0.0187

0.6523 0.4062 0.0820 0.0585

0.0089* 0.0258* 0.0060* 0.1295

*Significant P < 0.05. †After correction for multiple testing using the Hommel method.

of urinary oxidative stress metabolites allantoin and 2, 3 dinor-isoprostane F2-alpha correlated with distal pathway sterol levels (7-DHC and cholesterol) but not proximal sterols (Table 4). ALT levels were inversely correlated with oxidative stress markers, but no significant consistent relationship was observed for other liver enzymes. There were no significant differences in oxidative stress metabolite levels between HCV genotypes, SVR, or HOMA-IR levels.

Discussion In this pilot study we used a targeted metabolomics platform to better understand the interaction between the HCV virus and host lipid metabolism. To our knowledge, this is the first such analysis of these dedicated cholesterol pathway metabolites in patients with chronic HCV infection. Our patient cohort also provided the novel opportunity for comparison of sterol metabolites before and after treatment to account for host differences and better characterize the specific contribution of the virus to cholesterol metabolism. Using this approach, we demonstrated that HCV G3 infection lowers cholesterol compared to HCV G2. HCV genotype differences in cholesterol levels Table 4. Correlation Between Oxidative Stress Metabolites, Sterol Metabolites, and Liver Enzymes, All Genotypes (n532) Allantoin (mmol/mol)

Cholesterol 7-DHC Lathosterol Lanosterol ALT AST GGT

2,3 dinor-isopF2alpha (ng/mg)

iPF2alpha-VI (ng/mg)

R*

P

R*

P

R*

P

0.3112 0.3527 0.2257 0.1336 0.36 0.36 0.16

0.0830 0.0477 0.2142 0.4660 0.0444 0.0422 0.3953

0.3684 0.3642 0.0985 0.1415 0.35 0.11 0.08

0.0380 0.0404 0.5919 0.4398 0.0523 0.5551 0.6504

0.0187 0.0011 0.17 0.1616 0.40 0.27 0.23

0.9191 0.9952 0.3524 0.3771 0.0260 0.1351 0.2071

*Spearman correlation coefficient. 7-DHC, 7-dehydrocholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma glutamyltransferase.

have been observed previously.10 The assumption, therefore, was that the entire synthetic pathway should demonstrate similar differences by HCV genotype. Our analysis identified genotype differences in cholesterol, but not in the earlier precursor lanosterol (Fig. 2). When comparing the change in metabolite levels pre- versus posttreatment, in infected versus uninfected (SVR versus non-SVR), the differences were only apparent after lanosterol. This led to the hypothesis that the virus is associated with perturbation after the lanosterol step in the pathway, with a greater effect seen in G3 relative to G2. Our study provides no insight into a possible mechanism (e.g., direct viral interference by way of competitive inhibition between viral proteins and enzymes of sterol synthesis), but this is an important hypothesis to pursue experimentally in future research. Another alternative hypothesis is that HCV (especially G3) has no direct interference on cholesterol synthesis per se, but reduces cholesterol after it is synthesized. Why cholesterol precursors (lanosterol) are also not lower, and why host compensation does not overcome hypocholesterolemia, may be less well explained in the absence of viral-induced blockage in synthesis. Further subanalysis of several intermediate sterols did not identify a specific enzymatic process for HCV-targeted perturbation of the sterol pathway. For example, we found no similarities in the sterol pattern observed with HCV G3 to the metabolite patterns seen with enzyme dysfunction in well-characterized hereditary sterol disorders, such as 3b-hydroxysterol D7-reductase (Smith-Lemli-Opitz syndrome and desmosterolosis), 3b-hydroxysterol D24-reductase (desmosterolosis), 3bhydroxysteroid-D5-desaturase (lathosterolosis), and 3bhydroxysteroid-D8 D7-isomerase (X-Linked Dominant Conradi-Hu¨nermann syndrome; CDPX2).21,30 Eradication of HCV G3 resulted in increased distal metabolite levels but no change in the lanosterol level, further suggesting that viral perturbation of the sterol pathway may occur at a point(s) distal from lanosterol

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Fig. 2. Disruption of the cholesterol synthesis pathway in chronic HCV infection. The postsqualene sterol pathway is exclusively dedicated to cholesterol synthesis, which cannot occur except by way of these metabolites. HCV genotypic differences were found distally in the pathway with lower levels of cholesterol for HCV G3 compared to HCV G2 (Box 1). In HCV G3, patients who eradicated HCV after treatment had higher levels of distal metabolites lathosterol, 7-DHC, and cholesterol, but not of earlier sterol lanosterol, relative to those who did not clear virus (Box 2; see also Table 3). In contrast, no difference was found in lanosterol levels irrespective of whether HCV was present or absent (SVR versus non-SVR), and the lanosterol level did not differ by HCV genotype (G2 versus G3). Thus, HCV-related changes occur after lanosterol in the synthetic pathway. A low lanosterol level directly feeds-back to increase cholesterol synthesis by way of HMGCoA reductase, the rate-limiting step of cholesterol synthesis (broken line). Despite low cholesterol, in the absence of a low lanosterol level, no direct signal to reduce HMGCoA proteolysis is received. Distal pathway perturbation by HCV, with subsequent absence of the low lanosterol direct-feedback signal, could thus provide a hypothesis for the persistence of hypocholesterolemia frequently associated with chronic HCV infection.

and proximal to 7-DHC/cholesterol. Similar results were obtained when the changes before and after treatment metabolites were compared between SVR and non-SVR patients, and only postlanosterol metabolite changes differed with viral eradication. Additionally, higher G3 HCV RNA levels correlated with lower cholesterol, but this association was not apparent in G2. Our finding of HCV G3-specific perturbation of cholesterol metabolism is consistent with the clinical observations of increased prevalence of metabolic sequelae relative to other HCV genotypes, including more significant hypocholesterolemia, and increased rates and severity of hepatic steatosis5,10 that also inversely correlate with lower cholesterol for HCV

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G310 and differing effects on host metabolic gene expression.13 In keeping with our observation of an association between HCV RNA and low cholesterol in HCV G3, intrahepatic G3 HCV RNA has also been shown to correlate directly with other metabolic consequences of infection such as steatosis, further supporting a possible direct viral pathogenic effect.31 Oxidative stress in the endoplasmic reticulum has been linked to lipogenesis32 and HCV infection.33 In this small cohort, we found weak correlation between oxidative stress metabolites and sterol metabolites and ALT levels, but not in relation to genotype or IR. Further studies are required to characterize the role of these oxidative stress metabolites in HCV metabolic pathogenesis. An intriguing, yet unresolved question regarding HCV-host lipid interaction is: why hypocholesterolemia persists in HCV-infected patients, and is not overcome by normal host regulatory mechanisms to achieve cholesterol homeostasis? Our data suggest a possible hypothesis. HMGCoAR is the rate-limiting enzyme of cholesterol synthesis, yet surprisingly, low cholesterol levels provide only a relatively weak feedback effect on HMGCoAR.34 HMGCoAR is under highly complex regulation,35 but a critical regulator is feedback through the direct sterol-sensing of the lanosterol level (Fig. 1).26 Through a complex, endoplasmic reticulum-based process, low lanosterol signals to decrease proteolysis of HMGCoAR, which results in increased cholesterol synthesis.26,34 However, in the setting of normal lanosterol levels, such as observed in HCV infection, HMGCoAR proteolysis continues, and cholesterol synthesis is not up-regulated through this important pathway. Thus, with normal lanosterol levels, irrespective of cholesterol levels, the host fails to engage direct-sensing compensatory cholesterol synthesis through the rate-limiting step of HMGCoAR. This may facilitate the persistent hypocholesterolemia associated with HCV, a state which may also be favorable for viral replication.17 Pharmacological in vitro research suggests the distal sterol pathway is important for the HCV lifecycle and raises the potential for more selective inhibition of HCV replication by targeting this with small molecules and sterol antagonists.36,37 Our data would support this. Further targeted, in vitro studies are indicated to better identify the specific point(s) and nature of HCV interference in late cholesterol biosynthesis. Our analysis focused on the final part of the isoprenoid pathway that is exclusively dedicated to cholesterol synthesis. By restricting analysis to these dedicated cholesterol pathway steps (from lanosterol to cholesterol), we were able to exclude potential confounding flux in

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and out of the isoprenoid pathway from factors that are less dedicated to cholesterol synthesis, for example, involving earlier sterol metabolites or other nonsterol isoprenoids. Thus, by demonstrating no genotype or SVR-related differences in lanosterol levels, we controlled for any potential differences preceding this step in the presqualene mevalonate pathway. We were consequently unable to address the role of the earlier isoprenoid pathway steps and processes (such as geranylgeranylation14,38) that have been associated with viral replication, or alternative isoprenoid pathway products (such as prostaglandin and arachidonic acid). Importantly, however, we were able to address the late steps of cholesterol synthesis with a high degree of specificity, irrespective of possible earlier HCV interference in the sterol pathway. Despite the limitation of small sample size and correction for multiple testing, we have demonstrated significant differences when comparing patients infected with HCV G2 versus G3 genotypes at baseline, and resolution of these changes in HCV G3 after SVR. It may be that a similar, although attenuated, effect is apparent in non-G3 genotypes, and we were underpowered to see this smaller effect size. The absence of histopathological data from liver biopsy specimens is a limitation, but reflects current clinical practice for HCV G2/3 infection. This limits our ability to conclude that genotype differences in metabolites are linked to genotype differences in hepatic steatosis. However, linking serum and intrahepatic sterol pathway data to hepatic steatosis will be an important next step, particularly for patients with HCV G3 infection. Our results support further validation studies in a larger cohort, including patients infected with HCV G1, and available histological assessment of hepatic steatosis. In conclusion, using a novel targeted in vivo metabolomics approach in this pilot study we demonstrated that HCV G3 selectively interferes with cholesterol metabolism, resulting in lower distal metabolites, but preserved levels of the proximal sterol lanosterol. This interference resolves with HCV eradication. Based on the known feedback regulation mechanism of HMGCoAR, this sterol pattern of preserved lanosterol is likely to be permissive of hypocholesterolemia, and provides a hypothesis for the persistent hypocholesterolemia associated with HCV infection and its predominance in HCV G3. Our observations thus provide further insights into the genotype-specific relationship between virus and host cholesterol metabolism. These data also support the further investigation of distal host cholesterol metabolic pathways to better under-

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stand HCV-host lipid interactions in the viral lifecycle, disease pathogenesis, and as a potential adjunctive target for anti-HCV therapeutic research. Acknowledgment: Author contributions: P.J.C.: project concept and proposal, statistical analysis, interpretation, and wrote primary article; A.J.T.: project concept and proposal and article review; D.M.V.: statistical review and analysis and article review; L.E.K.: GC/MS, interpretation and article review; A.A.T.: LC/MSMS and article review, A.J.M.: article review; J.G.M.: article review; MS project concept and article review; D.M.M.: project proposal, LC/MSMS and article review; R.I.K.: GC/MS, interpretation and article review; K.P.: project concept, and proposal, interpretation and article review.

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Hepatitis C virus selectively perturbs the distal cholesterol synthesis ...

John G. McHutchison,1 Mani Subramanian,5 David M. Millington,4 Richard I. Kelley,3 and Keyur Patel1. Hepatitis C virus (HCV) subverts host cholesterol metabolism for key processes in ...... Ye J, Wang C, Sumpter R, Brown MS, Goldstein JL, Gale M. Disrup- tion of hepatitis C virus RNA replication through inhibition of host.

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