Hepatitis C virus production requires apolipoprotein A-I and affects its association with nascent low-density lipoproteins Carmine Mancone, 1 Corinna Steindler, 1 Laura Santangelo, 1,2 Giacoma Simonte, 1 Chrysoula Vlassi, 1 Maria Antonella Longo, 1 Gianpiero D’Offizi, 1 Cristina Di Giacomo, 1 Leopoldo Paolo Pucillo, 1 Laura Amicone, 2 Marco Tripodi, 1,2 Tonino Alonzi 1 ABSTRACT Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naı ¨ve HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy. INTRODUCTION Hepatitis C virus (HCV) has a major impact on public health with an estimated 170 million infected individuals worldwide, and is the primary reason for liver transplantation in Western Europe and the USA. Its infection causes chronic liver disease eventually leading to cirrhosis and hepato- cellular carcinoma. 1 Several abnormalities of lipid metabolism such as hypo-b-lipoproteinaemia and liver steatosis have also been associated with chronic HCV infection. 23 Many experimental studies have shown that the life cycle of HCV is directly linked to lipopro- teins. In the plasma of HCV-infected patients, the viral particles are associated with host lipids and apolipoproteins to form the so-called lipo-viro particles, mainly present in the range of very low- < Additional material is published online only. To view these files please visit the journal online (http://gut.bmj. com). 1 National Institute for Infectious Diseases L Spallanzani IRCCS, Rome, Italy 2 Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Universita ` Sapienza, Rome, Italy Correspondence to Tonino Alonzi, Laboratory of Gene Expression, National Institute for Infectious Diseases L Spallanzani IRCCS, Via Portuense 292, 00149 Rome, Italy; tonino.alonzi@inmi.it and Marco Tripodi, Dipartimento di Biotecnologie Cellulari ed Ematologia, Universita ` degli Studi di Roma “Sapienza”, Viale Regina Elena 324, 00161 Rome, Italy. tripodi@bce.uniroma1.it Revised 25 August 2010 Accepted 26 August 2010 Published Online First 12 October 2010 Significance of this study What is already known about this subject? < Chronic HCV infection is associated with major modifications of the host lipid metabolism. < The lipoprotein machinery is instrumental in several steps of the life cycle of HCV (ie, blood circulation, cell attachment and entry, viral particle assembly and release). < Circulating very low-density and low-density lipoproteins (VLDL and LDL) of HCV-infected patients cause alterations in the lipid metabo- lism of macrophages. What are the new findings? < Chronic HCV infection causes an impaired association of apolipoprotein A-I (apoA-I) with the circulating LDL particles of patients. < The impaired apoA-I/LDL association also occurs in hepatoma cells infected by HCV cell culture viral particles or expressing the sole viral non-structural proteins. < Impairment of the apoA-I/LDL association occurs during lipoprotein generation and is caused by the viral replication stage. < Downregulation of apoA-I induces significant impairment of HCV replication, showing that the replication stage of the viral life cycle also requires apolipoproteins. < Downregulation of apoA-I induces a significant decrease in HCV particle production in cell culture. How might it impact on clinical practice in the foreseeable future? < This study provides new insight into how HCV and the host lipoprotein machinery are recipro- cally influenced. < This evidence may result in new interest in this research area which may allow innovative antiviral strategies to be defined. 378 Gut 2011;60:378e386. doi:10.1136/gut.2010.211292 Hepatology group.bmj.com on December 12, 2016 - Published by http://gut.bmj.com/ Downloaded from