Circularization of an RNA template via long-range base pairing is critical for hepadnaviral reverse transcription

2009

J Virol

Self Cite

111

114

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...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Wang

Kim

2009

J Virol

Self Cite

111

114

“…The pT7 m -HBV construct that was used to generate the cap-free RNA was described previously (10). The HBV Pol-null construct that was made by an introduction of a frameshift mutation in the Pol gene was previously described (22). The plasmid constructs encoding eIF4E, eIF4E-W73A, and 4E-BP were the generous gift of N. Sonenberg (McGill University).…”

Section: Methodsmentioning

confidence: 99%

Incorporation of Eukaryotic Translation Initiation Factor eIF4E into Viral Nucleocapsids via Interaction with Hepatitis B Virus Polymerase

Kim

Wang

2010

J Virol

Self Cite

The DNA genome of hepatitis B virus (HBV) replicates via reverse transcription within capsids following the encapsidation of an RNA template, the pregenomic RNA (pgRNA). We previously demonstrated that the 5 cap proximity of the stem-loop structure ( or epsilon), an encapsidation signal, is critically important for the encapsidation of the pgRNA (J. K. Jeong, G. S. Yoon, and W. S. Ryu, J. Virol. 74:5502-5508, 2000). Therefore, we speculated that the viral polymerase (Pol), while bound to the 5 stem-loop structure, could recognize the cap via its interaction with eIF4E, a eukaryotic translation initiation factor. Our data showed the direct interaction between HBV Pol and eIF4E, as measured by coimmunoprecipitation. Further, we demonstrated that eIF4E interacts with the Pol-ribonucleoprotein complex (RNP) rather than Pol alone, resulting in eIF4E-Pol-RNP complex formation. In addition, we asked whether eIF4E remains engaged to the Pol-RNP complex during nucleocapsid assembly. Density gradient analysis revealed that eIF4E indeed was incorporated into nucleocapsids. It is of great importance to uncover whether the incorporated eIF4E contributes to viral reverse transcription or other steps in the HBV life cycle.Worldwide, an estimated 350 million persons are persistently infected with hepatitis B virus (HBV) (12). A significant subset of these HBV carriers progress to severe liver disease, such as hepatocellular carcinoma, which is assumed to cause up to one million deaths per year. Current treatment regimens for chronic HBV infections have limitations, so there is a clear medical need for new therapeutic strategies.HBV is a prototype of the hepadnavirus family, which includes duck hepatitis B virus and woodchuck hepatitis virus (21). Although they contain a DNA genome, hepadnaviruses replicate via the reverse transcription of an RNA template, the pregenomic RNA (pgRNA) (11). The pgRNA serves not only as the RNA template for viral reverse transcription but also as the mRNA that is used to encode two viral proteins required for viral genome replication: the core (capsid, or C) protein and the polymerase (Pol, or reverse transcriptase). It is not clear, however, whether and how the dual functions of the pgRNA are coordinated. Recently, we demonstrated that HBV Pol directs genome replication by suppressing the translation of the pgRNA (19). Therefore, we speculated that the translation suppression of the pgRNA could precede encapsidation. Importantly, the recognition of the 5Ј stem-loop structure (Fig. 1), called the encapsidation signal (ε or epsilon), by HBV Pol is critical for the translation suppression (19). In addition, the ability of Pol to specifically recognize the 5Ј ε also is critical for the initiation of viral reverse transcription as well as the assembly of replication-competent nucleocapsids (11,15,23). The epsilon sequence, initially defined as the RNA packaging signal that directs the specific encapsidation of the pgRNA into nucleocapsids, subsequently was found to be the origin of reverse transcriptio...

Insertion and Deletion Mutagenesis by Overlap Extension PCR

Lee

Shin

Methods in Molecular Biology

et al. 2010

Mutagenesis by the overlap extension PCR has become a standard method of creating mutations including substitutions, insertions, and deletions. Nonetheless, the established overlap PCR mutagenesis is limited in many respects. In particular, it has been difficult to make an insertion larger than 30 nt, since all sequence alterations must be embedded within the primer. Here, we describe a rapid and efficient method for creating insertions or deletions of any length at any position in a DNA molecule. This method is generally applicable, and therefore represents a significant improvement to the now widely used overlap extension PCR method.