Viruses utilize host factors in many steps of their life cycles. Yet, little is known about host factors that contribute to the life cycle of hepatitis B virus (HBV), which replicates its genome by reverse transcription. To identify host factors that contribute to viral reverse transcription, we sought to identify cellular proteins that interact with HBV polymerase (Pol) by using affinity purification coupled with mass spectrometry. One of the HBV Pol-interacting host factors identified was DDX3 DEAD-box RNA helicase, which unwinds RNA in an ATPase-dependent manner. Recently, it was shown that DDX3 is essential for both human immunodeficiency virus and hepatitis C virus infection. In contrast, we found that the ectopic expression of DDX3 led to significantly reduced viral DNA synthesis. The DDX3-mediated inhibition of viral DNA synthesis did not affect RNA encapsidation, a step prior to reverse transcription, and indicated that DDX3 inhibits HBV reverse transcription. Mutational analysis revealed that mutant DDX3 with an inactive ATPase motif, but not that with an inactive RNA helicase motif, failed to inhibit viral DNA synthesis. Our interpretation is that DDX3 inhibits viral DNA synthesis at a step following ATP hydrolysis but prior to RNA unwinding. Finally, OptiPrep density gradient analysis revealed that DDX3 was incorporated into nucleocapsids, suggesting that DDX3 inhibits viral reverse transcription following nucleocapsid assembly. Thus, DDX3 represents a novel host restriction factor that limits HBV infection.
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