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Abstract
The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from the LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ∼1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain and that mutations I853V, C2865F, and I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.
Highlights
Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases, including cirrhosis and hepatocellular carcinoma
After RNA transfection, the percentage of Core-positive cells was determined by immunostaining using anti-HCV Core antibody, and viral infectivity titers were determined by focus forming unit (FFU) assay as previously described [9]
The results showed that the spread ability of the TNcc was severely delayed in Huh7 cells compared with that in Huh7.5 cells, reaching peak infection (≥80% cell infected) at 62 and 14 days, respectively; the infectivity titer of Huh7-released TNcc virus was lower (103.2 FFU/ml) than that of Huh7.5 cell-released virus (104.0 FFU/ml) (Fig. 1, A and B)
Summary
Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases, including cirrhosis and hepatocellular carcinoma. The large number of undiagnosed patients and reinfections after cure and the limited accessibility and the unaffordability of direct-acting antiviral agent treatment in many underdeveloped countries and regions are delaying the global elimination of hepatitis C. Data from the World Health Organization show that annually 1.7 million people were infected by HCV from 2014 to 2018. Lipid droplets (LDs) and the endoplasmic reticulum (ER) are the sites for HCV envelopment to generate infectious mature virus particles [10, 11]. Orchestrated by viral and host proteins, mature Core proteins oligomerize in the ER and are translocated to the LDs, where viral RNAs are encapsidated to form nucleocapsids. The virus particles are further enveloped by apolipoproteins and very-low-density lipoprotein to gain infectivity and specific density [15, 16], and the particles with a buoyant density of 1.10 to 1.12 g/ml display the highest infectivity [6, 7, 17]
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