Elsevier

Journal of Hepatology

Volume 62, Issue 4, April 2015, Pages 763-770
Journal of Hepatology

Research Article
PCSK9, apolipoprotein E and lipoviral particles in chronic hepatitis C genotype 3: Evidence for genotype-specific regulation of lipoprotein metabolism

https://doi.org/10.1016/j.jhep.2014.11.016Get rights and content

Background & Aims

Hepatitis C virus (HCV) associates with lipoproteins to form “lipoviral particles” (LVPs) that can facilitate viral entry into hepatocytes. Initial attachment occurs via heparan sulphate proteoglycans and low-density lipoprotein receptor (LDLR); CD81 then mediates a post-attachment event. Proprotein convertase subtilisin kexin type 9 (PCSK9) enhances the degradation of the LDLR and modulates liver CD81 levels. We measured LVP and PCSK9 in patients chronically infected with HCV genotype (G)3. PCSK9 concentrations were also measured in HCV-G1 to indirectly examine the role of LDLR in LVP clearance.

Methods

HCV RNA, LVP (d <1.07 g/ml) and non-LVP (d >1.07 g/ml) fractions, were quantified in patients with HCV-G3 (n = 39) by real time RT-PCR and LVP ratios (LVPr; LVP/(LVP + non-LVP)) were calculated. Insulin resistance (IR) was assessed using the homeostasis model assessment of IR (HOMA-IR). Plasma PCSK9 concentrations were measured by ELISA in HCV-G3 and HCV-G1 (n = 51).

Results

In HCV-G3 LVP load correlated inversely with HDL-C (r = −0.421; p = 0.008), and apoE (r = −0.428; p = 0.013). The LVPr varied more than 35-fold (median 0.286; range 0.027 to 0.969); PCSK9 was the strongest negative predictor of LVPr (R2 = 16.2%; p = 0.012). HOMA-IR was not associated with LVP load or LVPr. PCSK9 concentrations were significantly lower in HCV-G3 compared to HCV-G1 (p <0.001). PCSK9 did not correlate with LDL-C in HCV-G3 or G1.

Conclusions

The inverse correlation of LVP with apoE in HCV-G3, compared to the reverse in HCV-G1 suggests HCV genotype-specific differences in apoE mediated viral entry. Lower PCSK9 and LDL concentrations imply upregulated LDLR activity in HCV-G3.

Introduction

Deaths from hepatitis C virus (HCV) have now superseded HIV as a cause of mortality in the United States and these deaths occur disproportionately in middle-aged persons [1]. HCV genotype 3 (HCV-G3) is the dominant genotype circulating in the UK and Southern Asia, and is most commonly found in European intravenous drug users [2]. Globally, it is estimated to account for 10–15% of the total number of HCV infections [3]. Chronic HCV-G3 patients have been found to have higher rates of late-stage liver disease and death [4], and HCV-G3 is now potentially the most difficult-to-treat genotype [5], most notably in these patients with decompensated cirrhosis.

HCV hijacks host lipid metabolism (reviewed in [6], [7]) leading to steatosis and hypocholesterolaemia, which resolves with successful HCV treatment [8]. One striking feature of infectious HCV particles is their buoyant density, which is unusually low for an RNA virus [9], due to interaction with lipoproteins [10]. In patients’ sera, HCV particles are found associated with very-low-density lipoprotein (VLDL) components (cholesterol, triglyceride, apolipoprotein (apo) B, apoE and apoCs), forming hybrid particles termed lipoviral particles (LVP). LVP can be immunoprecipitated with antibodies to apoB, apoE, and apoC1 [11], [12]. ApoE is enriched on infectious particles and correlates with infectivity [13]; electron microscopy studies have visualised apoE on the HCV envelope [14], [15]. Recent cryoelectron tomography studies have provided low-resolution 3D structural information on highly infectious virions and have shown that LVP incorporate apoB and apoA-I in addition to apoE [16].

Evidence has indicated that one function of HCV association with lipoproteins is the co-opting of lipoprotein receptors for attachment and entry into hepatocytes. The initial binding of LVP to cells occurs via interaction with low-density lipoprotein receptor (LDLR) and glycosaminoglycans present on heparan sulphate proteoglycans (HSPGs) [17] (reviewed in [18]). Both LDLR and HPSGs can interact with virion-associated apoE [19], [20]. CD81 then mediates a post-attachment event in HCV entry [21].

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a protease made by the liver. PCSK9 normally acts to enhance the degradation of the LDLR [22]. PCSK9 also modulates liver CD81 levels [23]. The circulating concentrations of human PCSK9 are directly correlated with LDL and total cholesterol concentrations in healthy human volunteers [24], [25]. Reducing the concentration or activity of PCSK9 enables greater numbers of LDLR on the cell surface, thereby increasing the clearance of LDL particles from the circulation and reducing plasma LDL cholesterol. LDLR binds and internalises LDL via its unique proteins, apoB100 and apoE.

We have previously measured low-density apoB-associated LVP in patients with chronic HCV-G1 and found a positive association with serum triglycerides, insulin resistance (IR) [26], and serum apoE levels [27]. However, there is evidence that virus-host interaction impacts on host lipid metabolism in ways, which may be HCV genotype-specific [28], [29], [30]. Therefore, we have, for the first time, examined LVP in the plasma of patients chronically infected with HCV-G3. We have measured PCSK9 concentrations in these patients and compared to patients chronically infected with HCV-G1 [26] to indirectly examine the role of LDLR in LVP clearance.

Section snippets

Ethics

The Newcastle upon Tyne Hospitals NHS Foundation Trust acted as sponsor and the study was approved by Northumberland Research Ethics Committee (REC number-07/H0902/45).

Patients

Patients with chronic hepatitis C (CHC), attending the viral hepatitis clinic at the Freeman Hospital, Newcastle upon Tyne and St Mary’s Hospital, Imperial College Healthcare Trust, London, were invited to participate and given a patient information leaflet explaining the study. Both treatment naïve and previous non-responders to

Clinical characteristics of the patients with chronic HCV-G3

The physical and metabolic characteristics of the 39 patients with CHC-G3 infection recruited in this study are summarised in Table 1 and fully detailed in Supplementary Table 1. Ethnicities were self-reported: Caucasian (n = 31), South Asian (n = 3), Middle Eastern (n = 2), East Asian (n = 1) and Mixed-other (n = 2; East/South Asian). BMI was normal (<25 kg/m2) in 17 patients, overweight (25–30 kg/m2) in 21 patients, and obese (>30 kg/m2) in 1 patient. According to the criteria of the International

Discussion

In chronic HCV infection, viral load is not a useful prognostic indicator of the severity of liver disease [35] and is influenced by a large number of demographic, viral, and human genetic factors [36]. In this study of HCV-G3 patients, we found that LDL-C was a significant determinant of total viral load; i.e., higher viral load, lower LDL-C. It has previously been shown that liver steatosis is independently associated with HCV-G3 [37] and that steatosis correlates with lower LDL-C and

Financial support

SHB, DAS, DJF, GLT, RDGN, and MFB were supported by the Medical Research Council (G0502028) and the Newcastle upon Tyne Healthcare Charity. HCT, MMEC, and SDT-R were supported by the NIHR Biomedical Facility at Imperial College London. NGS was supported by a CIHR grant and a Canada Chair. SHB is supported by an Anniversary Research Fellowship from Northumbria University.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Authors’ contributions

MFB, RDGN, HCT, SDT-R, and GLT conceived the study. SHB, DAS, DJF, MMEC, SDT-R, HCT, GLT, RDGN, and MFB were study investigators. DAS, MMEC, SDT-R, and MFB participated in the recruitment of patients and reporting of data for the enrolled patients. SHB, DAS, and DJF designed and performed experiments. SHB performed the statistical analysis. GD, NS, and JD contributed important reagents and collaborated with the study investigators. SHB, DAS, DJF, CVL, GLT, RDGN, and MFB contributed to the

Acknowledgements

SDT-R, MMEC, and HCT are grateful to the NIHR Biomedical Facility at Imperial College London for infrastructure support and to Claire Parsonage for assistance with biobanking facilities at Imperial College. We thank Pete Philipson for his statistical advice and the patients that participated in this study.

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