A Southern Blot Assay for Detection of Hepatitis B Virus Covalently Closed Circular DNA from Cell Cultures

Cai, Dawei; Nie, Hongtao; Yan, Ran; Guo, Ju‐Tao; Block, Timothy M.; Guo, Haitao

doi:10.1007/978-1-62703-484-5_13

Cited by 110 publications

(110 citation statements)

References 26 publications

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“…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). Briefly, protein-free vial DNAs were isolated from infected HepG2-NTCP cells in 35-mm plates (30).…”

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%

“…The extracted DNAs were treated with Plasmid-Safe ATP-dependent DNase according to the manufacturer's instructions (Epicentre). The viral DNAs were separated on an agarose gel, transferred to a nylon membrane, and hybridized with an HBV-specific probe, as described previously (26,31).…”

Section: Methodsmentioning

confidence: 99%

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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“…The cccDNA extracts were resolved on a 1.2% agarose gel in TAE (Tris-acetate-EDTA) buffer at 20 V for 16 h, as described before (41). The DNA was transferred overnight from the gel to a nylon membrane in 20ϫ SSC buffer (1ϫ SSC buffer is 0.15 M NaCl plus 0.015 M sodium citrate), using capillary force.…”

Section: Compoundsmentioning

confidence: 99%

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus

Berke

Dehertogh

Vergauwen

et al. 2017

Antimicrob Agents Chemother

114

126

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Hepatitis B virus (HBV) capsid assembly is a critical step in the propagation of the virus and is mediated by the core protein. Due to its multiple functions in the viral life cycle, core became an attractive target for new antiviral therapies. Capsid assembly modulators (CAMs) accelerate the kinetics of capsid assembly and prevent encapsidation of the polymerase-pregenomic RNA (Pol-pgRNA) complex, thereby blocking viral replication. CAM JNJ-632 is a novel and potent inhibitor of HBV replication in vitro across genotypes A to D. It induces the formation of morphologically intact viral capsids, as demonstrated by size exclusion chromatography and electron microscopy studies. Antiviral profiling in primary human hepatocytes revealed that CAMs prevented formation of covalently closed circular DNA in a dosedependent fashion when the compound was added together with the viral inoculum, whereas nucleos(t)ide analogues (NAs) did not. This protective effect translated into a dose-dependent reduction of intracellular HBV RNA levels as well as reduced HBe/cAg and HBsAg levels in the cell culture supernatant. The same observation was made with another CAM (BAY41-4109), suggesting that mechanistic rather than compound-specific effects play a role. Our data show that CAMs have a dual mechanism of action, inhibiting early and late steps of the viral life cycle. These effects clearly differentiate CAMs from NAs and may translate into higher functional cure rates in a clinical setting when given alone or in combination with the current standard of care.

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“…For total DNA from liver biopsies, approximately 2 μg total DNA was left untreated or coincubated with T5 exonuclease or topoisomerase I (New England BioLabs) and loaded on 1.2% agarose gel. For detection of cccDNA, after electrophoresis, gels were depurinated by 0.2 M hydrochloride, followed by alkaline denaturation and neutralization essentially as described previously (42). DNA was transferred onto a positively charged nylon membrane (Roche Life Science) using a vacuum blotter (Bio-Rad).…”

Section: Localization Of Hbcag In Clinical Hbv Infectionmentioning

confidence: 99%

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Zhang¹,

Lu²,

Zheng³

et al. 2016

Journal of Clinical Investigation

106

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A Southern Blot Assay for Detection of Hepatitis B Virus Covalently Closed Circular DNA from Cell Cultures

Cited by 110 publications

References 26 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

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

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