Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

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.

Cell-Cell Contact-Mediated Hepatitis C Virus (HCV) Transfer, Productive Infection, and Replication and Their Requirement for HCV Receptors

“…Contrary to our studies, other studies have found that nocodazole treatment inhibits microtubule-dependent transport of the HCV replication complex, initiation of productive HCV infection, and, as a result, HCV RNA replication (75,77,78). Moreover, two recent reports have shown that postassembly the vesicular HCV core protein puncta traffic via microtubules and that trafficking is inhibited by nocodazole treatment (28,50). It is important to point out that all the above-mentioned studies used much higher concentrations of nocodazole that were mostly 10 to 20 times higher than the concentration that we used in our studies.…”

“…We therefore turned to the TC tag-biarsenical dye labeling system that has successfully been used to label and study several viruses in live cells (reviewed in reference 49), including HCV (28,50). Compared to the GFP-core protein fusion protein strategy, this system requires insertion of only a short nucleotide sequence encoding a peptide of 12 amino acids, including the tetracysteine (TC) tag (-CCXXCC-), into the viral genome without affecting virus translation, replication, assembly, production, and infectivity (28,49,50). The presence of the TC tag allows the tagged HCV core protein in the cells or in HCV to be detected live by microscopic imaging through its covalent binding to the cell membrane-permeant nonfluorescent biarsenical compound and its ensuing fluorophore.…”

“…When the RNA genome from the replication complex on the membranous web and the envelope proteins (E1/E2) on the ER are complete, viral assembly/envelopment takes place on the ER outer membrane in close proximity to the lipid droplets, toward the ER lumen (50). These intracellular viruses become infectious upon envelopment and are thought to be loaded into secretory vesicles that egress via the ER-trans Golgi network secretory pathway and are released from the plasma membrane (28). However, the fusion of HCV-loaded vesicles with the plasma membrane (the actual release process) has never been captured with fluorescence or electron microscopy.…”

Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

“…It should be noted that the Golgi and endocytic pathways intersect and the exit routes from the trans-Golgi network (TGN) also include the early, late, and recycling endosomes (15,16). A recent study applying siRNA screening highlights the importance of ER-Golgi trafficking in HCV secretion, as well as lipid and protein kinases that function in TGN cargo vesicle budding (17). Rab11A, a small GTP binding protein that modulates trafficking from recycling endosomes and the Golgi and PI4KIII␤, a PI4-kinase localized to the Golgi were shown to be required for HCV virion secretion (17).…”

Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes

“…Rab11a-positive recycling endosomes sort some vesicles to define the directions during exocytosis (41,42). Hepatitis C virus particles are localized in recycling endosomes transiently and the recycling endosomes serve as a sorting station (43). We found that WNV particles colocalized with Rab11-and transferrin-positive recycling endosomes in infected cells.…”