DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

Abstract

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.Viruses rely on host factors to complete their life cycles. These factors facilitate many steps of the viral life cycle, including entry, uncoating, genome replication, viral assembly, and virus release (3, 13). Recently, some host factors that contribute to the life cycles of some clinically important human viruses were identified by full-genome small interfering RNA knockdown experiments (7,14,31). For instance, nearly 300 host factors that contribute to human immunodeficiency virus (HIV) infection were identified (7). Yet, little is known about host factors that contribute to the genome replication of hepatitis B virus (HBV).HBV, the prototypical member of the hepadnavirus family, is a major cause of liver disease worldwide (34). HBV-mediated disease manifestations range from acute and chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (HCC). Although HBV contains a DNA genome, the replication of the genome occurs by reverse transcription of the pregenomic RNA (pgRNA) template. HBV polymerase (Pol), or reverse transcriptase, acts as an RNA binding protein by specifically recognizing an RNA stem-loop structure called the 5Ј ε encapsidation signal (5Ј ε), and this interaction is required for pgRNA encapsidation (5, 15, 17). Viral reverse transcription occurs entirely within nucleocapsids following encapsidation. HBV reverse transcription has two steps for DNA synthesis: (i) minus-strand DNA synthesis and (ii) plus-strand DNA synthesis. During the first step, the pgRNA is converted i...

DDX19A Senses Viral RNA and Mediates NLRP3-Dependent Inflammasome Activation

“…These results demonstrate that DDX19A specifically functions in HP-PRRSV RNA-induced IL-1b secretion by acting as the direct sensor, and other stimulators (such as polyI:C or ATP/LPS), respectively, activate NLRP3 inflammasome through different pathways. Recently, the DEAD/H-box helicase family members have been drawing more and more attention not only for their powerful capacities in detecting invading PAMPs as direct sensors (6, 36), but also for their key roles involved in viral replication (36,38,39,71). It has been demonstrated that DDX3 binds the HCV core FIGURE 8.…”

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

“…One prerequisite for a cellular protein to be subjected to such a modification would likely be a close interaction with RT so that it can gain access to the RT active site. The RT protein is known to interact with a number of cellular proteins, including molecular chaperone proteins (13,15), the helicase DDX3 (52), and the translation factor eIF4E (22). The RT protein is also thought to be cytotoxic when overexpressed and has been reported to affect cellular functions such as interferon signaling and gene expression (2,8,14,53).…”

Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation

“…Replication of the genome occurs by reverse transcription of the pregenomic RNA template, is mediated by the HBV polymerase which binds to an RNA stem loop and occurs entirely within nucleocapsids. It was recently shown that DDX3 binds to HBV polymerase, in an interaction that did not appear to be mediated by RNA [43]. DDX3 was incorporated into nucleocapsids together with HBV polymerase and inhibited the initial step of reverse transcription in a manner that seemed to 25 depend on the ATPase-activity of DDX3 [43].…”

“…It was recently shown that DDX3 binds to HBV polymerase, in an interaction that did not appear to be mediated by RNA [43]. DDX3 was incorporated into nucleocapsids together with HBV polymerase and inhibited the initial step of reverse transcription in a manner that seemed to 25 depend on the ATPase-activity of DDX3 [43]. A lot of questions remain unanswered, such as the exact mechanism of inhibition and the physiological relevance of the finding.…”

“…A lot of questions remain unanswered, such as the exact mechanism of inhibition and the physiological relevance of the finding. It is interesting that DDX3 is targeted by two hepatitis viruses, raising the question as to whether targeting DDX3 might contribute to liver cell tropism [43]. However, HCV and HBV belong to different virus families and have different replication mechanisms, making a common strategy for targeting DDX3 unlikely.…”