Dynamic Interaction of Stress Granules, DDX3X, and IKK-α Mediates Multiple Functions in Hepatitis C Virus Infection

The ubiquitous ATP-dependent RNA helicase DDX3X is involved in many cellular functions, including innate immunity, and is a pivotal host factor for hepatitis C virus (HCV) infection. Recently, we showed that DDX3X specifically recognizes the HCV 3' untranslated region (UTR), leading to the activation of IKK-alpha and a cascade of lipogenic signaling to facilitate lipid droplet biogenesis and viral assembly (Q. Li, V. Pene, S. Krishnamurthy, H. Cha, and T. J. Liang, Nat Med 19:722-729, 2013, http://dx.doi.org/10.1038/nm.3190). The interaction of DDX3X with HCV core protein seems to be dispensable for its proviral role. In this study, through systematic imaging and biochemical and virologic approaches, we identified a dynamic association between DDX3X and various cellular compartments and viral elements mediating multiple functions of DDX3X in productive HCV infection. Upon HCV infection, the HCV 3' UTR interacts with DDX3X and IKK-alpha, which redistribute to speckle-like cytoplasmic structures shown to be stress granules (SGs). As viral proteins accumulate in infected cells, DDX3X granules together with SG-associated proteins redistribute and colocalize with HCV core protein around lipid droplets (LDs). IKK-alpha, however, does not relocate to the LD but translocates to the nucleus. In HCV-infected cells, various HCV nonstructural proteins also interact or colocalize with DDX3X in close proximity to SGs and LDs, consistent with the tight juxtaposition of the replication complex and the assembly site at the surface of LDs. Short interfering RNA (siRNA)-mediated silencing of DDX3X and multiple SG components markedly inhibits HCV infection. Our data suggest that DDX3X initiates a multifaceted cellular program involving dynamic associations with HCV RNA and proteins, IKK-alpha, SG, and LD surfaces for its crucial role in the HCV life cycle. IMPORTANCE DDX3X is a proviral host factor for HCV infection. Recently, we showed that DDX3X binds to the HCV 3' UTR, activating IKK-alpha and cellular lipogenesis to facilitate viral assembly (Q. Li et al., Nat Med 19:722-729, 2013, http://dx.doi.org/10.1038/nm.3190). Here, we report associations of DDX3X with various cellular compartments and viral elements that mediate its multiple functions in the HCV life cycle. Upon infection, the HCV 3' UTR redistributes DDX3X and IKK-alpha to speckle-like cytoplasmic structures shown to be SGs. Subsequently, interactions between DDX3X, SG, and HCV proteins facilitate the translocation of DDX3X-SG complexes to the LD surface. HCV nonstructural proteins are shown to colocalize with DDX3X in close proximity to SGs and LDs, consistent with the tight juxtaposition of the HCV replication complex and assembly site at the LD surface. Our data demonstrate that DDX3X initiates a multifaceted cellular program involving dynamic associations with HCV elements, IKK-alpha, SGs, and LDs for its critical role in HCV infection.