Abstract
Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.
Highlights
HCV3 infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma
Because of the chimeric nature of the Jc1 genome, we assumed that the higher titer and faster kinetics of virus production must be due to determinants residing in the J6 sequence
We initially focused on the core protein and constructed J6/JFH1 chimeras as follows: one analogous to Jc1 but encoding the core gene of the JFH1 isolate (JFH1core/Jc1), and another containing the J6 core coding region in the context of the JFH1 genome (Fig. 1A, J6core/JFH1)
Summary
Because of high permissiveness for HCV RNA replication upon transfection and superior properties for immunofluorescence microscopy, most experiments were performed with Huh7-Lunet cells. Samples were centrifuged at 10,000 ϫ g for 10 min at 4 °C to remove cell debris Infectivity titers of these lysates and cell culture supernatants were determined by using limited dilution assay on Huh7.5 cells as described recently [27, 29]. After washing with PBS and blocking with PBS containing 5% normal goat serum, cells were incubated with primary antibody diluted in PBS/goat serum for 1 h at room temperature. Blots were washed three times for 10 min in washing solution (0.5% Tween 20 in PBS), incubated for 1 h with the horseradish peroxidase-conjugated secondary antibody in blocking solution, and washed as described above. The normalized level of fluorescence was averaged and plotted against time
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