Abstract
Cytosolic lipid droplets are central organelles in the Hepatitis C Virus (HCV) life cycle. The viral capsid protein core localizes to lipid droplets and initiates the production of viral particles at lipid droplet–associated ER membranes. Core is thought to encapsidate newly synthesized viral RNA and, through interaction with the two envelope proteins E1 and E2, bud into the ER lumen. Here, we visualized the spatial distribution of HCV structural proteins core and E2 in vicinity of small lipid droplets by three-color 3D super-resolution microscopy. We observed and analyzed small areas of colocalization between the two structural proteins in HCV-infected cells with a diameter of approximately 100 nm that might represent putative viral assembly sites.
Highlights
Hepatitis C virus (HCV) persistently infects,180 million people worldwide and the associated morbidity and mortality are a major public health concern [1]
Lipid droplets can be visualized in dSTORM To visualize lipid droplets and surrounding structures by superresolution microscopy we first analyzed different lipophilic fluorescent dyes (BODIPY, LipidTox Green, LipidTox Red) for their ability to blink upon excitation
To verify that the signals detected in dSTORM represent lipid droplets we correlated widefield fluorescence microscopy of LipidTox Red stained Huh7 Lunet cells with the corresponding dSTORM image
Summary
Hepatitis C virus (HCV) persistently infects ,180 million people worldwide and the associated morbidity and mortality are a major public health concern [1]. HCV is a single positive-stranded RNA virus of the Flaviviridae family that is translated into a single polyprotein upon entry into host cells. Host and viral proteases cleave this polyprotein, releasing the 10 individual viral proteins. The structural proteins, the viral capsid core and the envelope glycoproteins E1 and E2, are the components of virions while the nonstructural proteins NS3–5B form the viral RNA replication complex. The establishment of fully permissive cell culture systems (HCVcc) [2,3] revealed a close connection between host cell lipids and HCV replication at each step of the viral replication cycle reviewed in [4]. The cellular storage organelles of lipids, lipid droplets, emerged as putative viral assembly sites [5,6,7]
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