Abstract
ABSTRACTHepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread.
Highlights
Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission
We initially screened for interactions between the adaptor proteins (APs)-1A, AP-1B, AP-2, and AP-4 complexes and the HCV proteome by using protein fragment complementation assays (PCAs)
Since the subunits of AP complexes interact with both tyrosine and dileucine motifs [31,32,33,34], their coding genes were fused to an N-terminal luciferase fragment reporter (GLuc1-A), while individual HCV proteins derived from the J6/JFH genome [44] were fused to an N-terminal complementary luciferase fragment (GLuc2-B)
Summary
Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. IMPORTANCE HCV spreads via cell-free infection or cell-to-cell contact that shields it from antibody neutralization, thereby facilitating viral persistence Factors governing this differential sorting remain unknown. We reveal that AP-4, an adaptor not previously implicated in viral infections, mediates cell-to-cell spread and HCV trafficking. HCV particles, rendered infectious upon budding, exit the cell via the secretory pathway [7], where they cotraffic with various components of the ER, the trans-Golgi network (TGN), and recycling endosomes [8] Upon their release, these cell-free viral particles can infect distant cells. Cell-to-cell spread is implicated in immune evasion, HCV persistence, and antiviral treatment failure [15, 16] It remains unknown, how viral particles are differentially directed to cell membrane sites for cell-to-cell versus cell-free spread
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