1140 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF ADIPOSE DIFFERENTIATION-RELATED PROTEIN IN LIPID DROPLET MORPHOLOGY AND VIRAL LIFE CYCLE

ABSTRACTHepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction.IMPORTANCEHepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

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Role of seipin in lipid droplet morphology and hepatitis C virus life cycle

Journal of General Virology ◽

10.1099/vir.0.054593-0 ◽

2013 ◽

Vol 94 (10) ◽

pp. 2208-2214 ◽

Cited By ~ 6

Author(s):

Sophie Clément ◽

Catherine Fauvelle ◽

Emilie Branche ◽

Vincent Kaddai ◽

Stéphanie Conzelmann ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Droplet ◽

Outer Surface ◽

Lipid Droplets ◽

Particle Production ◽

Critical Factor ◽

Infectious Hepatitis ◽

Particle Assembly ◽

Droplet Morphology

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

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Osteopontin Regulates Hepatitis C Virus (HCV) Replication and Assembly by Interacting with HCV Proteins and Lipid Droplets and by Binding to Receptors αVβ3 and CD44

Journal of Virology ◽

10.1128/jvi.02116-17 ◽

2018 ◽

Vol 92 (13) ◽

pp. e02116-17 ◽

Cited By ~ 6

Author(s):

Jawed Iqbal ◽

Mehuli Sarkar-Dutta ◽

Steven McRae ◽

Akshaya Ramachandran ◽

Binod Kumar ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Droplets ◽

Critical Role ◽

Migration And Invasion ◽

Core Proteins ◽

Important Relationship ◽

Infected Cells

ABSTRACT Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVβ3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVβ3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVβ3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly. IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVβ3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

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The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control

Journal of Hepatology ◽

10.1016/j.jhep.2016.07.035 ◽

2016 ◽

Vol 65 (1) ◽

pp. S2-S21 ◽

Cited By ~ 98

Author(s):

Jens Bukh

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Basic Research ◽

Viral Life Cycle ◽

Epidemic Control ◽

History Of

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A184 DISSECTING THE ROLE OF THE POLY(C)-BINDING PROTEIN 2 IN THE HEPATITIS C VIRUS LIFE CYCLE

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwy008.185 ◽

2018 ◽

Vol 1 (suppl_1) ◽

pp. 320-320

Author(s):

S Cousineau ◽

S Sagan

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Binding Protein ◽

Virus Life Cycle

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Intracellular Hepatitis C Virus Modeling Predicts Infection Dynamics and Viral Protein Mechanisms

Journal of Virology ◽

10.1128/jvi.02098-17 ◽

2018 ◽

Vol 92 (11) ◽

pp. e02098-17 ◽

Cited By ~ 4

Author(s):

Thomas R. Aunins ◽

Katherine A. Marsh ◽

Gitanjali Subramanya ◽

Susan L. Uprichard ◽

Alan S. Perelson ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Kinetic Data ◽

Viral Proteins ◽

Viral Life Cycle ◽

Hcv Infection ◽

Viral Rna ◽

Model Parameters ◽

Virus Life Cycle

ABSTRACTHepatitis C virus (HCV) infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these infections becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral life cycle. Here, we present a detailed mathematical model that represents the full hepatitis C virus life cycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses ofcis- andtrans-acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady state. Our model predicts higher rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the life cycle.IMPORTANCEWe have designed a model for the full life cycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C virus replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded it to create a model that incorporates the full virus life cycle as well as additional intracellular mechanistic detail. We compared several different hypotheses that have been proposed for different parts of the life cycle and applied the corresponding model variations to infection data to determine which hypotheses are most consistent with the empirical kinetic data. Because the infection data we have collected for this study are a more physiologically relevant representation of a viral life cycle than data obtained from a replicon system, our model can make more accurate predictions about clinical hepatitis C virus infections.

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