Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis

Ko, Chunkyu; Shin, Younchul; Park, Woojin; Kim, Seungtaek; Kim, Jong-Hwa; Ryu, Wang Shick

doi:10.1128/jvi.01916-13

Cited by 26 publications

(24 citation statements)

References 30 publications

(29 reference statements)

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“…Cells were transfected with polyethylenimine (PEI) (linear,25 kDa; Polysciences, Inc.) as previously described (29). For lentiviral production, HEK293T cells were seeded in 100-mm plates and transfected as described below.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, one-third of the RNA isolated from a 35-mm plate was subjected to an RNase protection assay as recommended by the manufacturer (Ambion). The pgRNA-specific riboprobe was generated as described previously (29). A riboprobe for the detection of gylceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was also generated by in vitro transcription of the GAPDH gene fragment.…”

Section: Methodsmentioning

confidence: 99%

“…Southern blot analysis was performed using viral DNAs isolated from cytoplasmic capsids, exactly as previously described (29). For the detection of cccDNA, a modified Hirt method was used to extract protein-free vial DNAs, as described previously (26,30,31).…”

Section: Methodsmentioning

confidence: 99%

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DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level

Lee

Windisch

et al. 2014

J Virol

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DDX3 is a member of the DEAD-box RNA helicase family, involved in mRNA metabolism, including transcription, splicing, and translation. We previously identified DDX3 as a hepatitis B virus (HBV) polymerase (Pol) binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at the posttranscriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed a diverse HBV experimental system. Inconsistently, we found that DDX3-mediated inhibition occurs at the level of transcription. By using tetracycline-inducible HBV-producing cells, we observed that lentivirus-mediated DDX3 expression led to a reduced level of HBV RNAs. Importantly, knockdown of DDX3 by short hairpin RNA resulted in augmentation of HBV RNAs in two distinct HBV replication systems: (i) tetracyclineinducible HBV-producing cells and (ii) constitutive HBV-producing HepG2.2.15 cells. Moreover, DDX3 knockdown in HBVsusceptible HepG2-NTCP cells, where covalently closed circular DNA (cccDNA) serves as the template for viral transcription, resulted in increased HBV RNAs, validating that transcription regulation by DDX3 occurs on a physiological template. Overall, our results demonstrate that DDX3 represents an intrinsic host antiviral factor that restricts HBV transcription. IMPORTANCE Upon entry into host cells, viruses encounter host factors that restrict viral infection. During evolution, viruses have acquired the ability to subvert cellular factors that adversely affect their replication. Such host factors include TRIM5␣ and APOBEC3G, which were discovered in retroviruses. The discovery of host restriction factors provided deeper insight into the innate immune response and viral pathogenesis, leading to better understanding of host-virus interactions. In contrast to the case with retroviruses, little is known about host factors that restrict hepatitis B virus (HBV), a virus distantly related to retroviruses. DDX3DEAD box RNA helicase is best characterized as an RNA helicase involved in RNA metabolism, such as RNA processing and translation. Here, we show that DDX3 inhibits HBV infection at the level of viral transcription. C hronic hepatitis B virus (HBV) infection represents a major public health burden, affecting more than 300 million individuals worldwide, and carries a high risk for developing cirrhosis and hepatocellular carcinoma (HCC) (1). HBV virions contain a small, partially double-stranded circular DNA genome of 3.2 kb in length. Although it is a DNA virus, HBV replicates its DNA genome via reverse transcription. Upon infection, the virion DNA is converted into covalently closed circular DNA (cccDNA), which then serves as the template for viral transcription (2). Among the viral transcripts, only pregenomic RNA (pgRNA), 3.5 kb in length, is selectively packaged into nucleocapsids along with HBV polymerase (Pol). Inside the nucleocapsid, the pgRNA is reverse transcribed by HBV Pol to yield relaxed circular (RC) DNA. These mature RC DNA-containin...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level

Lee

Windisch

et al. 2014

J Virol

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“…Southern blot analysis showed that R703A, D777A and R781A mutants significantly reduced amounts of viral DNAs, which revealed that three charged residues of the HBV polymerase RNaseH domain contribute to the catalysis of RNaseH in removing the RNA template, but not in the RNA encapsidation [133,134]. HBV reverse transcription requires the viral RNase H to destroy the viral RNA after it has been translated into minus strand DNA.…”

Section: Inhibitors Of Viral Dna Synthesismentioning

confidence: 98%

Recent Advance of the Hepatitis B Virus Inhibitors: A Medicinal Chemistry Overview

et al. 2015

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Hepatitis B Virus (HBV) is one of the most prevalent viral infections of human worldwide. The therapies are limited in the clinical context because of negative side effects of interferons and the development of viral resistance to the nucleoside/nucleotide inhibitors. In this review, we summarize the recent advances in design and development of potent anti-HBV inhibitors from natural sources and synthetic compounds, targeting different steps in the life cycle of HBV. We attempt to emphasize the major structural modifications, mechanisms of action and computer-aided docking analysis of novel potent inhibitors that need to be addressed in the future to design potent anti-HBV molecules.

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“…Southern blot analysis of HBV DNA replication intermediates was performed as previously described (23). Briefly, 4 days after transfection, cells were harvested, and viral DNA was isolated from cytoplasmic capsids.…”

Section: Methodsmentioning

confidence: 99%

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms

Kim

Lee

Son

et al. 2016

J Virol

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HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx. IMPORTANCEDDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms. H epatitis B virus (HBV) infection represents a major global public health concern, with over 300 million chronically infected patients worldwide. Chronic HBV infection carries a great risk of developing into severe liver diseases, including cirrhosis and liver cancer, which result in a million deaths annually (1). HBV carries a small (ca. 3.2-kb), relaxed circular (RC), partially double-stranded DNA. Following entry into the host cell, the viral RC DNA traffics to the nucleus, where it is converted into a covalently closed circular DNA (cccDNA), the template for viral transcription (2). Four viral RNAs are transcribed from their respective promoters and serve a...

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Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis

Cited by 26 publications

References 30 publications

DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level

DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level

Recent Advance of the Hepatitis B Virus Inhibitors: A Medicinal Chemistry Overview

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms

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