Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is an important public health problem. Polygonum perfoliatum L. is a traditional medicinal herb and has been reported to have pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. In this study, the antiviral activities and mechanisms of Polygonum perfoliatum L. extract against HBV and the effective components were investigated. The results showed that the total extract of Polygonum perfoliatum L. reduced the levels of HBV e antigen (HBeAg) secretion and the viral covalently closed circular DNA (CCC DNA) formation, but had little or no negative effects on viral capsid assembly and pregenomic RNA packaging. Further fractionation showed that the water extract (WE) fraction exerted comparable anti-HBV activities with the total extract, especially in inhibiting the CCC DNA formation and HBeAg production, indicating that the effective antiviral components are mainly distributed in this fraction. Further study showed that the phenolic acids constituents, protocatechuic acid, and gallic acid, but not ethyl caffeate, which is reported enriched in the WE fraction, showed strong anti-HBV activities in inhibiting viral core DNA synthesis, CCC DNA formation, and HBeAg production. These results suggested that the Polygonum perfoliatum L. total extract and the related phenolic acids like protocatechuic acid and gallic acid could inhibit HBV replication and also indicated the potential utility of Polygonum perfoliatum L. and related constituents as sources of novel antivirals against HBV.

Discovery of a small-molecule inhibitor targeting the ovarian tumor domain of a novel Tamdy orthonairoviruse associated with human febrile illness.

Tick-borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV-1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor (OTU) domain of TcTV-1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV-1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV-1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by the TcTV-1 virus.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Switching Field Distribution in BN/FePtCAg/MgTiON and FePtCAg/MgTiOBN Films.

BN is the currently required segregant for perpendicular FePt media. We found that BN can be diffused from the MgTiOBN intermediate layer during a high temperature process. The FePtCAg film sputtered on MgTiOBN layers illustrates higher perpendicular magnetocrystalline anisotropy (Ku) (1.43 × 107 erg/cm3) and coercivity (normal to film surface) (17 kOe) at 350 K compared to BN/FePtCAg/MgTiON film. From the microstructure, the FePtCAg film shows the granular structure on the MgTiOBN intermediate layer, but parts of the irregular FePt grains are agglomerated and partially separated in the matrix, with grains size being, on average, 26.7 nm. Cross-sectional imaging showed that the FePt grains have a truncated pyramid shape with a lower wetting angle, which is influenced by the surface energy of MgTiOBN. BN segregation at FePt grains or boundaries is still not clear. Using the electron energy loss spectrum (EELS), we found that part of the BN atoms were clearly observed in the FePt lattice and iron-boride oxide was indexed in the x-ray photoelectron spectroscopy (XPS) spectra. To determine the effects of BN segregant (from capping layer or intermediate layer) on the magnetic switching behavior of FePtCAg film, the intrinsic-(ΔHint = 6.17 kOe, 6.54 kOe) and extrinsic- (ΔHext = 0.80 kOe, 0.39 kOe) switching field distribution (SFD) were measured by plotting saturated major- and unsaturated minor- hysteresis loops to evaluate the crystal orientation and microstructure (grains volume and distribution) for BN/FePtCAg/MgTiON and FePtCAg/MgTiOBN films, respectively. The main contribution of intrinsic SFD is the c-axis misalignment for the BN/FePt/MgTiON sample; however, the dispersed magnetic anisotropy has a higher input to intrinsic SFD for FePtCAg/MgTiOBN/CrRu film.

Inhibitory activities of Ranunculus japonicus Thunb. ethanol extract against hepatitis B virus.

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation.

Urinary neonicotinoid insecticides in children from South China: Concentrations, profiles and influencing factors.

Neonicotinoid insecticides can selectively interact with the unique nicotinic acetylcholine receptor subtypes in insects and are considered to be low toxic to mammals. However, there is still insufficient knowledge on human exposure to neonicotinoid insecticides, especially for children. This study aimed to investigate urinary concentrations and profiles of neonicotinoid insecticides in South China children and to analyze potential influencing factors. Six neonicotinoid insecticides, including imidacloprid (IMI), thiamethoxam (THM), acetamiprid (ACE), clothianidin (CLO), thiacloprid (THD) and dinotefuran (DIN), exhibited high detection frequencies (>90%) in urine samples collected from 305 children, suggesting broad exposure in South China children. The median concentrations were determined to be 0.13, 0.21, 0.01, 0.19, 0.002 and 1.64 µg/L, respectively. Among the target neonicotinoids, urinary concentrations of CLO and THM exhibited a significant and positive correlation between each other (p < 0.05), suggesting similar sources of these two chemicals.

TBC1D15 affects glucose uptake by regulating GLUT4 translocation.

Glucose transport into skeletal muscle is mediated by the principal glucose transporter protein, GLUT4, which can be transported from intracellular vesicles to the cytoplasmic membrane, and the translocation of GLUT4 vesicles requires a variety of Rab proteins. Previously we reported a new type of TBC1D15 from C. plagiosum with Rab-GAP activity for Rab7. Here we reported that TBC1D15 regulated glucose uptake by affecting the translocation of GLUT4 through late endosomal pathway. When TBC1D15 was knocked out by CRISPR/Cas9, a significant reduction in 2-NBDG uptake was observed, and the total amount of GLUT4 was significantly reduced in TBC1D15-/- cells compared to that in WT cells. Furthermore, concentrated distribution of Rab7 in Lamp1-decorated late endosome/lysosome and an increase in co-localization between GLUT4 and Rab7 was observed in TBC1D15-/- cells. These results suggested that TBC1D15 served as a master regulator in GLUT4 translocation and further affected GLUT4-mediated glucose uptake.

HIV-HBV and HIV-HCV Coinfection and Liver Cancer Development.

Liver diseases that are caused by the hepatitis B virus (HBV) and hepatitis C virus (HCV), including cirrhosis and hepatocellular carcinoma (HCC), have become increasingly important in patients infected with the human immunodeficiency virus (HIV) as their life expectancy is getting longer with successful anti-HIV therapy. Due to their shared transmission routes, dual infection by HIV and HBV or HIV and HCV, and triple infection by all three viruses are fairly common and affect millions of people worldwide. Whereas the immunodeficiency caused by HIV enhances the likelihood of HBV and HCV persistence, hepatotoxicity associated with anti-HIV therapy can worsen the liver diseases associated with HBV or HCV persistence. Evidence suggests HIV infection increases the risk of HBV- or HCV-associated HCC risk although the precise mechanisms of enhanced hepatocarcinogenesis remain to be fully elucidated. Recent success in curing HCV infection, and the availability of therapeutic options effective in long-term suppression of both HIV and HBV replication, bring hope, fortunately, to those who are coinfected but also highlight the need for judicious selection of antiviral therapies.

Bmo-miR-79 downregulates the expression of BmEm4 in the silkworm, Bombyx mori.

MicroRNA is an important regulation factor in insect development and metamorphosis. It has been reported that E(spl)m4 is a miRNA-targeted gene, as well as the target of the Notch signaling pathway in Drosophila. The expression of E(spl)m4 can be regulated by microRNA and further affect the neural development of Drosophila. Here, we found that BmEm4, an ortholog of E(spl)m4 from Bombyx mori, was the target gene of bmo-miR-79, with target sites containing the Brd and K boxes of the BmEm4_3'UTR, which was validated by the dual luciferase reporter (DLR) assay. Furthermore, bmo-miR-79 mimics can inhibit the expression of BmEm4 in BmN cells after transfection, and bmo-miR-79 can also inhibit the expression of BmEm4 in different developmental stages of Bombyx mori at a posttranscriptional level, to different degrees. The EMSA test further showed that bmo-miR-79 could bind to BmAGO2, which is the Bombyx mori argonaute2 protein, suggesting that bmo-miR-79 might regulate the expression of BmEm4 by forming miRISC complexes with BmAGO2. Taken together, bmo-miR-79 could regulate the expression of BmEm4 mediated by BmAGO2 and further affect its function in the silkworm Bombyx mori.

1α,25-Dihydroxyvitamin D3 inhibits aflatoxin B1-induced proliferation and dedifferentiation of hepatic progenitor cells by regulating PI3K/Akt and Hippo pathways.

Hepatic progenitor cells (HPCs) might be the origin of hepatocellular carcinoma. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) (VD3) has been documented as an anticancer agent for various cancers. However, the potential effect of VD3 on the proliferation and malignant transformation of HPCs induced by aflatoxin B1 (AFB1) has not been determined. In this study, we found that AFB1 exhibited the stimulative effects on the proliferation, dedifferentiation and invasion of HPCs via activating AKT pathway but turning off Hippo pathway, which were terminated when VD3 was used in combination with AFB1. Furthermore, in AFB1-induced liver damage mouse model, VD3 also showed protective effect by reducing HPCs population. Together, these preclinical data not only provide a newly identified mechanism by which AFB1 affects HPCs but also strengthen the idea of developing VD3 as an anticancer agent.

Multiple roles of core protein linker in hepatitis B virus replication.

Hepatitis B virus (HBV) core protein (HBc) contains an N-terminal domain (NTD, assembly domain) and a C-terminal domain (CTD), which are linked by a flexible linker region. HBc plays multiple essential roles in viral replication, including capsid assembly, packaging of the viral pregenomic RNA (pgRNA) into nucleocapsids, viral reverse transcription that converts pgRNA to the genomic DNA, and secretion of DNA-containing (complete) virions or genome-free (empty) virions. The HBc linker is generally assumed to act merely as a spacer between NTD and CTD but some results suggest that the linker may affect NTD assembly. To determine its role in viral replication, we have made a number of deletion and substitution mutants in the linker region, in either the presence or absence of CTD, and tested their abilities to support capsid assembly and viral replication in human cells. Our results indicate that the linker could indeed impede NTD assembly in the absence of CTD, which could be partially relieved by partial linker deletion. In contrast, when CTD was present, the linker deletions or substitutions did not affect capsid assembly. Deletion of the entire linker or its C-terminal part resulted in a partial defect in pgRNA packaging and severely impaired viral DNA synthesis. In contrast, deletion of the N-terminal part of the linker, or substitutions of the linker sequence, had little to no effect on RNA packaging or first-strand DNA synthesis. However, the N-terminal linker deletion and two linker substitution mutants were defective in the production of mature double-stranded viral DNA. Secretion of empty virions was blocked by all the linker deletions and substitutions tested. In particular, a conservative linker substitution that allowed mature viral DNA synthesis and secretion of complete virions severely impaired the secretion of empty virions, thus increasing the ratio of complete to empty virions that were secreted. Together, these results demonstrate that the HBc linker region plays critical and complex roles at multiple stages of HBV replication.

Isolation and characterization of Bacillus sp. GFP-2, a novel Bacillus strain with antimicrobial activities, from Whitespotted bamboo shark intestine.

The abuse of antibiotics and following rapidly increasing of antibiotic-resistant pathogens is the serious threat to our society. Natural products from microorganism are regarded as the important substitution antimicrobial agents of antibiotics. We isolated a new strain, Bacillus sp. GFP-2, from the Chiloscyllium plagiosum (Whitespotted bamboo shark) intestine, which showed great inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria. Additionally, the growth of salmon was effectively promoted when fed with inactivated strain GFP-2 as the inhibition agent of pathogenic bacteria. The genes encoding antimicrobial peptides like LCI, YFGAP and hGAPDH and gene clusters for secondary metabolites and bacteriocins, such as difficidin, bacillibactin, bacilysin, surfactin, butirosin, macrolactin, bacillaene, fengycin, lanthipeptides and LCI, were predicted in the genome of Bacillus sp. GFP-2, which might be expressed and contribute to the antimicrobial activities of this strain. The gene encoding ß-1,3-1,4-glucanase was successfully cloned from the genome and this protein was detected in the culture supernatant of Bacillus sp. GFP-2 by the antibody produced in rabbit immunized with the recombinant ß-1,3-1,4-glucanase, indicating that this strain could express ß-1,3-1,4-glucanase, which might partially contribute to its antimicrobial activities. This study can enhance a better understanding of the mechanism of antimicrobial activities in genus Bacillus and provide a useful material for the biotechnology study in antimicrobial agent development.

Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System.

The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.

Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-Free Hepatitis B Virions.

During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (i) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (ii) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required but could stimulate empty virion secretion. Also, substitutions in L that eliminated secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that blocked secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids and mature nucleocapsids interact with the S and L proteins during the formation of complete and empty virions.IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases, including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV-infected cells also secrete noninfectious, incomplete viral particles in large excess over the number of complete virions. In particular, the empty (or genome-free) virion shares with the complete virion the outer envelope and interior capsid but contains no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis and have implications for efforts to develop empty HBV virions as novel biomarkers and a new generation of HBV vaccine.

Bivariate Correlation Analysis of the Chemometric Profiles of Chinese Wild Salvia miltiorrhiza Based on UPLC-Qqq-MS and Antioxidant Activities.

To better understand the mechanisms underlying the pharmacological actions of Salvia miltiorrhiza, correlation between the chemical profiles and in vitro antioxidant activities in 50 batches of wild S. miltiorrhiza samples was analyzed. Our ultra-performance liquid chromatography-tandem mass spectrometry analysis detected twelve phenolic acids and five tanshinones and obtained various chemical profiles from different origins. In a principal component analysis (PCA) and cluster analysis, the tanshinones cryptotanshinone, tanshinone IIA and dihydrotanshinone I exhibited higher weights in PC1, whereas the phenolic acids danshensu, salvianolic acids A and B and lithospermic acid were highly loaded in PC2. All components could be optimized as markers of different locations and might be suitable for S. miltiorrhiza quality analyses. Additionally, the DPPH and ABTS assays used to comprehensively evaluate antioxidant activities indicated large variations, with mean DPPH and ABTS scavenging potencies of 32.24 and 23.39 µg/mL, respectively, among S. miltiorrhiza extract solutions. Notably, samples that exceeded the mean IC50 values had higher phenolic acid contents. A correlation analysis indicated a strong correlation between the antioxidant activities and phenolic acid contents. Caffeic acid, danshensu, rosmarinic acid, lithospermic acid and salvianolic acid B were major contributors to antioxidant activity. In conclusion, phenolic compounds were the predominant antioxidant components in the investigated plant species. These plants may be sources of potent natural antioxidants and beneficial chemopreventive agents.

Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application.

Hepatitis B virus (HBV) is a para-retrovirus or retroid virus that contains a double-stranded DNA genome and replicates this DNA via reverse transcription of a RNA pregenome. Viral reverse transcription takes place within a capsid upon packaging of the RNA and the viral reverse transcriptase. A major characteristic of HBV replication is the selection of capsids containing the double-stranded DNA, but not those containing the RNA or the single-stranded DNA replication intermediate, for envelopment during virion secretion. The complete HBV virion particles thus contain an outer envelope, studded with viral envelope proteins, that encloses the capsid, which, in turn, encapsidates the double-stranded DNA genome. Furthermore, HBV morphogenesis is characterized by the release of subviral particles that are several orders of magnitude more abundant than the complete virions. One class of subviral particles are the classical surface antigen particles (Australian antigen) that contain only the viral envelope proteins, whereas the more recently discovered genome-free (empty) virions contain both the envelope and capsid but no genome. In addition, recent evidence suggests that low levels of RNA-containing particles may be released, after all. We will summarize what is currently known about how the complete and incomplete HBV particles are assembled. We will discuss briefly the functions of the subviral particles, which remain largely unknown. Finally, we will explore the utility of the subviral particles, particularly, the potential of empty virions and putative RNA virions as diagnostic markers and the potential of empty virons as a vaccine candidate.

Capsid Phosphorylation State and Hepadnavirus Virion Secretion.

The C-terminal domain (CTD) of hepadnavirus core protein is involved in multiple steps of viral replication. In particular, the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging into immature nucleocapsids (NCs) and the early stage of viral DNA synthesis. For the avian hepadnavirus duck hepatitis B virus (DHBV), CTD is dephosphorylated subsequently to facilitate the late stage of viral DNA synthesis and to stabilize NCs containing mature viral DNA. The role of CTD phosphorylation in virion secretion, if any, has remained unclear. Here, the CTD from the human hepatitis B virus (HBV) was found to be dephosphorylated in association with NC maturation and secretion of DNA-containing virions, as in DHBV. In contrast, the CTD in empty HBV virions (i.e., enveloped capsids with no RNA or DNA) was found to be phosphorylated. The potential role of CTD dephosphorylation in virion secretion was analyzed through mutagenesis. For secretion of empty HBV virions, which is independent of either viral RNA packaging or DNA synthesis, multiple substitutions in the CTD to mimic either phosphorylation or dephosphorylation showed little detrimental effect. Similarly, phospho-mimetic substitutions in the DHBV CTD did not block the secretion of DNA-containing virions. These results indicate that CTD dephosphorylation, though associated with NC maturation in both HBV and DHBV, is not essential for the subsequent NC-envelope interaction to secrete DNA-containing virions, and the CTD state of phosphorylation also does not play an essential role in the interaction between empty capsids and the envelope for secretion of empty virions.IMPORTANCE The phosphorylation state of the C-terminal domain (CTD) of hepatitis B virus (HBV) core or capsid protein is highly dynamic and plays multiple roles in the viral life cycle. To study the potential role of the state of phosphorylation of CTD in virion secretion, we have analyzed the CTD phosphorylation state in complete (containing the genomic DNA) versus empty (genome-free) HBV virions. Whereas CTD is unphosphorylated in complete virions, it is phosphorylated in empty virions. Mutational analyses indicate that neither phosphorylation nor dephosphorylation of CTD is required for virion secretion. These results demonstrate that while CTD dephosphorylation is associated with HBV DNA synthesis, the CTD state of phosphorylation may not regulate virion secretion.

Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation.

UNLABELLED: Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE: Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes.

UNLABELLED: Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response that, in turn, modulates viral gene expression and replication. IMPORTANCE: Chronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can, under select conditions, be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence.

Regulation of multiple stages of hepadnavirus replication by the carboxyl-terminal domain of viral core protein in trans.

UNLABELLED: Mutational analyses have indicated that the carboxyl-terminal domain (CTD) of hepadnavirus core protein and its state of phosphorylation are critical for multiple steps in viral replication. Also, CTD interacts with host proteins in a phosphorylation state-dependent manner. To ascertain the role of CTD in viral replication without perturbing its sequence and the role of CTD-host interactions, CTD of the human hepatitis B virus (HBV) or duck hepatitis B virus (DHBV) core protein, either the wild type (WT) or with alanine or glutamic acid/aspartic acid substitutions at the phosphorylation sites, was expressed in cells replicating DHBV with the WT core protein. A dramatic decrease in phosphorylation of the DHBV core protein (DHBc) was observed when the WT and most HBV core protein CTD (HCTD) variants were coexpressed in trans, which was accompanied by a profound reduction of viral core DNA and, in particular, the double-stranded DNA. One HCTD variant that failed to change DHBc phosphorylation also had no effect on DHBV core DNA. All WT and variant HCTDs and DHBc CTDs (DCTDs) decreased the DHBV covalently closed circular (CCC) DNA. Identification of CTD-host interactions indicated that CDK2 binding by CTD may mediate its inhibitory effect on DHBc phosphorylation and reverse transcription via competition with DHBc for the host kinase, whereas importin α binding by CTD may contribute to inhibition of CCC DNA production by competitively blocking the nuclear import of viral nucleocapsids. These results suggest the possibility of blocking multiple steps of viral replication, especially CCC DNA formation, via inhibition of CTD functions. IMPORTANCE: Mutational analyses have suggested that the carboxyl-terminal domain (CTD) of hepadnavirus core protein is critical for viral replication. However, results from mutational analyses are open to alternative interpretations. Also, how CTD affects virus replication remains unclear. In this study, we took an alternative approach to mutagenesis by overexpressing CTD alone in cells replicating the virus with the wild-type core protein to determine the roles of CTD in viral replication. Our results revealed that CTD can inhibit multiple stages of viral replication, and its effects may be mediated at least in part through specific host interactions. They suggest that CTD, or its mimics, may have therapeutic potential. Furthermore, our experimental approach should be broadly applicable as a complement to mutagenesis for studying protein functions and interactions while at the same time providing a means to identify the relevant interacting factors.