Proximity between the cap and 5′ ε stem–loop structure is critical for the suppression of pgRNA translation by the hepatitis B viral polymerase

Ryu, Dong Kyun; Ahn, Byung Yoon; Ryu, Wang Shick

doi:10.1016/j.virol.2010.07.005

Cited by 17 publications

(13 citation statements)

References 21 publications

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“…3, lanes 7, 11, and 25). This result was consistent with previous reports (22,43,44) and could be explained by the known inhibitory effect of P on pgRNA translation due to the occlusion of ribosomes from pgRNA upon P protein-RNA interaction. Other than the effects of the polymerase mutations on core protein expression, some variations in transfection efficiency might have contributed to the variation of core expression.…”

Section: Figsupporting

confidence: 94%

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase

Clark

Flanagan

2017

J Virol

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Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain. IMPORTANCE HBV is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. One important feature of this virus is its polymerase, the enzyme used to create the DNA genome from a specific viral RNA by reverse transcription. One region of this polymerase, the TP domain, is required for association with the viral RNA and production of the DNA genome. Targeting the TP domain for antiviral development is difficult due to the lack of homology to other proteins and highresolution structure. This study mapped the TP functions according to predicted secondary structure, where it folds into alpha helices or unstructured loops. Three predicted loops were found to be the most important regions functionally and the most conserved evolutionarily. Identification of these functional subdomains in TP will facilitate its targeting for antiviral development.

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Section: Figsupporting

confidence: 94%

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase

Clark

Flanagan

2017

J Virol

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“…60 nucleotides from the 5′ end cap, very similar to pgRNA packaging requirement. 69,91 The mechanism by which HP suppresses translation is not entirely clear, but it is thought that HP binding to the 5′ Hε could prevent ribosomal binding and subsequent translation. It is also unresolved why the position of the 5′ Hε is critical for both RNA packaging and translational repression, although it could be related to cap binding factors that require a short enough distance to interact with and regulate the HP–Hε complex for both activities.…”

Section: Suppression Of Pgrna Translationmentioning

confidence: 99%

Hepatitis B virus reverse transcriptase: diverse functions as classical and emerging targets for antiviral intervention

Jones¹,

2013

Emerging Microbes & Infections

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Hepatitis B virus (HBV) infection remains a global health problem with over 350 million chronically infected, causing an increased risk of cirrhosis and hepatocellular carcinoma. Current antiviral chemotherapy for HBV infection include five nucleos(t)ide analog reverse transcriptase inhibitors (NRTIs) that all target one enzymatic activity, DNA strand elongation, of the HBV polymerase (HP), a specialized reverse transcriptase (RT). NRTIs are not curative and long-term treatment is associated with toxicity and the emergence of drug resistant viral mutations, which can also result in vaccine escape. Recent studies on the multiple functions of HP have provided important mechanistic insights into its diverse roles during different stages of viral replication, including interactions with viral pregenomic RNA, RNA packaging into nucleocapsids, protein priming, minus- and plus-strand viral DNA synthesis, RNase H-mediated degradation of viral RNA, as well as critical host interactions that regulate the multiple HP functions. These diverse functions provide ample opportunities to develop novel HP-targeted antiviral treatments that should contribute to curing chronic HBV infection.

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“…Intracellular viral core DNA was extracted as described previously (22). For isolation of cccDNAs, protein-free viral DNAs were extracted from HBV-infected cells with the Hirt method, with minor modifications (21).…”

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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Proximity between the cap and 5′ ε stem–loop structure is critical for the suppression of pgRNA translation by the hepatitis B viral polymerase

Cited by 17 publications

References 21 publications

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase

Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase

Hepatitis B virus reverse transcriptase: diverse functions as classical and emerging targets for antiviral intervention

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

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