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Hepatitis 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. IMPORTANCE HCV spreads via cell-free infection or cell-to-cell contact that shields it from antibody neutralization, thereby facilitating viral persistence. Yet, factors governing this differential sorting remain unknown. By integrating proteomic, RNA interference, genetic, live-cell imaging, and pharmacological approaches, we uncover differential coopting of host adaptor proteins (APs) to mediate HCV traffic at distinct late steps of the viral life cycle. We reported that AP-1A and AP-2 mediate HCV trafficking during release and assembly, respectively. Here, we demonstrate that dileucine motifs in the NS2 protein mediate AP-1A, AP-1B, and AP-4 binding and cell-free virus release. Moreover, we reveal that AP-4, an adaptor not previously implicated in viral infections, mediates cell-to-cell spread and HCV trafficking. Lastly, we demonstrate cell-to-cell spread regulation by AAK1 and GAK, host kinases controlling APs, and susceptibility to their inhibitors. This study provides mechanistic insights into virus-host determinants that facilitate HCV trafficking, with potential implications for pathogenesis and antiviral agent design.

Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release