Abstract
The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.
Highlights
Hepatitis C virus (HCV) is a member of the Flaviviridae family that contains a single-stranded positive RNA genome (,9600bp)
The current model of HCV egress is that virions assemble at lipid droplets, envelope at the endoplasmic reticulum (ER) and likely exit the hepatocyte via the secretory pathway in association with apolipoproteins
To gain a more detailed insight into infectious HCV release, we combined an RNA interference (RNAi) analysis of host factors that are required for infectious HCV secretion with live cell imaging of HCV core trafficking
Summary
Hepatitis C virus (HCV) is a member of the Flaviviridae family that contains a single-stranded positive RNA genome (,9600bp). This is translated into a polyprotein, which is processed by viral and host proteases into 3 structural proteins (core and the glycoproteins, E1 and E2) and 7 non-structural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). The discovery of HCV strains that are infectious in cell culture has enhanced the appreciation of the functions of these proteins in all stages of the viral life cycle [1,2,3,4]. Infection of hepatocytes in cell culture requires at least four potential receptor molecules, CD81, SR-B1, claudins, and occludin [5,6,7,8]. Replication is associated membranous structures derived from the endoplasmic reticulum (ER) called the membranous web [12,13]
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