Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA.

Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

A yellow fever virus NS4B inhibitor not only suppresses viral replication, but also enhances the virus activation of RIG-I-like receptor-mediated innate immune response.

Flavivirus infection of cells induces massive rearrangements of the endoplasmic reticulum (ER) membrane to form viral replication organelles (ROs) which segregates viral RNA replication intermediates from the cytoplasmic RNA sensors. Among other viral nonstructural (NS) proteins, available evidence suggests for a prominent role of NS4B, an ER membrane protein with multiple transmembrane domains, in the formation of ROs and the evasion of the innate immune response. We previously reported a benzodiazepine compound, BDAA, which specifically inhibited yellow fever virus (YFV) replication in cultured cells and in vivo in hamsters, with resistant mutation mapped to P219 of NS4B protein. In the following mechanistic studies, we found that BDAA specifically enhances YFV induced inflammatory cytokine response in association with the induction of dramatic structural alteration of ROs and exposure of double-stranded RNA (dsRNA) in virus-infected cells. Interestingly, the BDAA-enhanced cytokine response in YFV-infected cells is attenuated in RIG-I or MAD5 knockout cells and completely abolished in MAVS knockout cells. However, BDAA inhibited YFV replication at a similar extent in the parent cells and cells deficient of RIG-I, MDA5 or MAVS. These results thus provided multiple lines of biological evidence to support a model that BDAA interaction with NS4B may impair the integrity of YFV ROs, which not only inhibits viral RNA replication, but also promotes the release of viral RNA from ROs, which consequentially activates RIG-I and MDA5. Although the innate immune enhancement activity of BDAA is not required for its antiviral activity in cultured cells, its dual antiviral mechanism is unique among all the reported antiviral agents thus far and warrants further investigation in animal models in future.

Long-term antiviral therapy is associated with changes in the profile of transcriptionally active HBV integration in the livers of patients with CHB.

Hepatitis B virus (HBV) integration exists throughout the clinical course of chronic hepatitis B (CHB). This study investigated the effects of long-term antiviral therapy on the level and profiles of transcriptionally active HBV integration. Serial liver biopsies and paired blood samples were obtained from 16, 16, and 22 patients with CHB at baseline, 78, and 260 weeks of entecavir monotherapy or combined with pegylated interferon alfa, respectively. Serum HBV biomarkers were longitudinally assessed. RNA-seq and HIVID2 program was used to identify HBV-host chimeric RNAs transcribed from integrated DNA. The counts of HBV integration reads were positively related to both serum HBV DNA levels (r = 0.695, p = 0.004) and HBeAg titers (r = 0.724, p = 0.021) at baseline, but the positive correlation exited only to the serum HBsAg levels after 260 weeks of antiviral therapy (r = 0.662, p = 0.001). After 78 weeks of antiviral therapy, the levels of HBV integration expression decreased by 12.25 folds from baseline. The viral junction points were enriched at the S and HBx genes after the long-term antiviral therapy. HBs-FN1 became one of the main transcripts, with the mean proportion of HBs-FN1 in all integrated expression increased from 2.79% at baseline to 10.54% at Week 260 of antiviral treatment. Antiviral therapy may reduce but not eliminate the HBV integration events and integration expression. Certain integration events, such as HBs-FN1 can persist in long-term antiviral treatment.

Association between kidney stones and life's essential 8: a population-based study.

BACKGROUND: Kidney stones exhibit a robust correlation with cardiovascular disease (CVD). The objective of this research is to investigate the correlation between kidney stones and Life's Essential 8 (LE8), a newly updated assessment of cardiovascular health (CVH), among adults in the United States. METHODS: In this study, which analyzed data from the 2007-2018 National Health and Nutrition Examination Survey, we employed LE8 scores (ranging from 0 to 100) as the independent variable, classifying them into low, moderate, and high CVH categories. The research examined the relationship between LE8 scores and kidney stones by using multivariate logistic regression and restricted cubic spline models, with kidney stones as the dependent variable. RESULTS: Out of the 14,117 participants in this research, the weighted mean LE8 score was 69.70 ± 0.27. After accounting for confounding factors, there was an inverse association between higher LE8 scores and the likelihood of developing kidney stones (OR of 0.81 per 10-point increase, with a 95% confidence interval of 0.77-0.85), demonstrating a non-linear dose-response pattern. Similar patterns were observed for health behaviors, health factor scores, and kidney stones. Stratified analyses demonstrated a stable negative correlation between LE8 scores and kidney stones across different subgroups. CONCLUSION: LE8 and its subscale scores exhibited a robust and inverse correlation with the occurrence of kidney stones. Encouraging adherence to optimal CVH levels has the potential to serve as an effective strategy in preventing and minimizing the occurrence of kidney stones.

RNA-binding MSI proteins and their related cancers: A medicinal chemistry perspective.

Musashi1 and Musashi2 are RNA-binding proteins originally found in drosophila, in which they play a crucial developmental role. These proteins are pivotal in the maintenance and differentiation of stem cells in other organisms. Research has confirmed that the Musashi proteins are highly involved in cell signal-transduction pathways such as Notch and TGF-ß. These signaling pathways are related to the induction and development of cancers, such as breast cancer, leukemia, hepatoma and liver cancer. In this review we focus on how Musashi proteins interact with molecules in different signaling pathways in various cancers and how they affect the physiological functions of these pathways. We further illustrate the status quo of Musashi proteins-targeted therapies and predict the target RNA regions that Musashi proteins interact with, in the hope of exploring the prospect of the design of Musashi protein-targeted medicines.

A cross-sectional analysis of the relationship between the non-high density to high density lipoprotein cholesterol ratio (NHHR) and kidney stone risk in American adults.

BACKGROUND: Recent interest in the Non-High Density to High Density Lipoprotein Cholesterol ratio (NHHR) has emerged due to its potential role in metabolic disorders. However, the connection between NHHR and the development of kidney stones still lacks clarity. The primary goal of this research is to explore how NHHR correlates with kidney stone incidence. METHODS: An analysis was conducted on the data collected by the National Health and Nutrition Examination Survey (NHANES) between 2007 and 2018, focusing on adults over 20 years diagnosed with kidney stones and those with available NHHR values. Employing weighted logistic regression and Restricted Cubic Spline (RCS) models, NHHR levels' correlation with kidney stone risk was examined. Extensive subgroup analyses were conducted for enhanced reliability of the findings. RESULTS: The findings indicate a heightened kidney stone risk for those at the highest NHHR levels relative to those at the lowest (reference group). A notable non-linear correlation of NHHR with kidney stone incidence has been observed, with a significant P-value (< 0.001), consistent across various subgroups. CONCLUSION: A clear link exists between high NHHR levels and increased kidney stone risk in the American adult population. This study highlights NHHR's significance as a potential indicator in kidney stone formation.

Synthesis and Application of Salicylhydrazone Probes with High Selectivity for Rapid Detection of Cu2.

Using the aldehyde amine condensation procedure and the triphenylamine group as the skeleton structure, the new triphenylamine-aromatic aldehyde-succinylhydrazone probe molecule DHBYMH was created. A newly created acylhydrazone probe was structurally characterized by mass spectrometry (MS), NMR, and infrared spectroscopy (FTIR). Fluorescence and UV spectroscopy were used to examine DHBYMH's sensing capabilities for metal ions. Notably, DHBYMH achieved a detection limit of 1.62 × 10-7 M by demonstrating exceptional selectivity and sensitivity towards Cu2+ ions in an optimum sample solvent system (DMSO/H2O, (v/v = 7/3); pH = 7.0; cysteine (Cys) concentration: 1 × 10-4 M). NMR titration, high-resolution mass spectrometry analysis, and DFT computation were used to clarify the response mechanism. Ultimately, predicated on DHBYMH's reversible identification of Cu2+ ions in the presence of EDTA, a molecular logic gate was successfully designed.

Androgen receptor and MYC transcriptomes are equilibrated in multilayer regulatory circuitries in prostate cancer.

BACKGROUND: The discovery of androgen receptor (AR) having transrepression effects completes the circle of its functionalities as a typical transcription factor, which intrinsically bears dual functions of activation and repression linked to co-factor competition and redistribution. Indeed, AR dual functions are exemplified by locus-wide regulation of the oncogenic 8q24-MYC region. METHODS: RT-qPCR assay and public RNA-profiling datasets were used to assess MYC transcription in androgen-sensitive cell lines. Public ChIP-seq and RNA-Seq datasets were computed to evaluate AR-MYC direct and indirect signatures. Gene sets in typical MYC and AR pathways were monitored to validate their cross-talks. Bio-informatics and chromosome conformation capture (3C) assay were performed in the AR gene locus to examine androgen-elicited distal regulation. Finally, co-factor re-distribution were globally tracked between AR and MYC binding sites. RESULTS: In this report, we found MYC responded negatively to androgen with hypersensitivity, rivaling AR natural functions as an innate androgen effector. Furthermore, both direct and indirect AR and MYC transcriptional programs were actively in equilibration. With established androgen-mediated versus MYC-mediated gene subsets, we validated AR and MYC pathways were both bidirectional and extensively entangled. In addition, we determined that the AR gene locus resembled the MYC gene region and both loci were androgen-repressed via epigenetics and chromatin architectural alterations. Significantly, transcriptional factor profiling along the prostate cancer (PCa) genome exposed that PCa transcriptomes were dynamically equilibrated between AR-binding site and MYC-binding site. CONCLUSION: Together, our findings stratified AR-MYC interactions that are extensively wired and intricately organized to compensate for essential PCa transcriptional programs and neutralize excessive signaling.

Pregenomic RNA Launch Hepatitis B Virus Replication System Facilitates the Mechanistic Study of Antiviral Agents and Drug-Resistant Variants on Covalently Closed Circular DNA Synthesis.

Hepatitis B virus (HBV) replicates its genomic DNA by reverse transcription of an RNA intermediate, termed pregenomic RNA (pgRNA), within nucleocapsid. It had been shown that transfection of in vitro-transcribed pgRNA initiated viral replication in human hepatoma cells. We demonstrated here that viral capsids, single-stranded DNA, relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA) became detectable sequentially at 3, 6, 12, and 24 h post-pgRNA transfection into Huh7.5 cells. The levels of viral DNA replication intermediates and cccDNA peaked at 24 and 48 h post-pgRNA transfection, respectively. HBV surface antigen (HBsAg) became detectable in culture medium at day 4 posttransfection. Interestingly, the early robust viral DNA replication and cccDNA synthesis did not depend on the expression of HBV X protein (HBx), whereas HBsAg production was strictly dependent on viral DNA replication and expression of HBx, consistent with the essential role of HBx in the transcriptional activation of cccDNA minichromosomes. While the robust and synchronized HBV replication within 48 h post-pgRNA transfection is particularly suitable for the precise mapping of the HBV replication steps, from capsid assembly to cccDNA formation, targeted by distinct antiviral agents, the treatment of cells starting at 48 h post-pgRNA transfection allows the assessment of antiviral agents on mature nucleocapsid uncoating, cccDNA synthesis, and transcription, as well as viral RNA stability. Moreover, the pgRNA launch system could be used to readily assess the impacts of drug-resistant variants on cccDNA formation and other replication steps in the viral life cycle. IMPORTANCE Hepadnaviral pgRNA not only serves as a template for reverse transcriptional replication of viral DNA but also expresses core protein and DNA polymerase to support viral genome replication and cccDNA synthesis. Not surprisingly, cytoplasmic expression of duck hepatitis B virus pgRNA initiated viral replication leading to infectious virion secretion. However, HBV replication and antiviral mechanism were studied primarily in human hepatoma cells transiently or stably transfected with plasmid-based HBV replicons. The presence of large amounts of transfected HBV DNA or transgenes in cellular chromosomes hampered the robust analyses of HBV replication and cccDNA function. As demonstrated here, the pgRNA launch HBV replication system permits the accurate mapping of antiviral target and investigation of cccDNA biosynthesis and transcription using secreted HBsAg as a convenient quantitative marker. The effect of drug-resistant variants on viral capsid assembly, genome replication, and cccDNA biosynthesis and function can also be assessed using this system.

Dissecting transcription of the 8q24-MYC locus in prostate cancer recognizes the equilibration between androgen receptor direct and indirect dual-functions.

BACKGROUND: Androgen receptor (AR) activation and repression dual-functionality only became known recently and still remains intriguing in prostate cancer (PCa). MYC is a prominent oncogene that functionally entangles with AR signaling in PCa. Further exploration of AR regulatory mechanisms on MYC gene transcription bears clinical and translation significance. METHODS: Bioinformatics analysis of PCa cell line and clinical RNA-Seq and ChIP-Seq (chromatin immunoprecipitation-sequencing) datasets to anchor interactions of AR and MYC transcriptional networks. ChIP-qPCR and 3C (chromosome conformation capture) analyses to probe MYC distal regulation by AR binding sites (ABSs). CRISPR/Cas9-mediated genome-editing to specify functions of ABS within the 8q24-MYC locus on androgen-mediated MYC transcription. Global FoxA1 and HoxB13 distribution profiling to advance AR transcriptional mechanisms. RESULTS: Here we recognize AR bi-directional transcription mechanisms by exploiting the prominent 8q24-MYC locus conferring androgen hyper-sensitivity. At ~ 25 Kb downstream of the MYC gene, we identified an undefined ABS, P10. By chromatin analyses, we validated androgen-dependent spatial interaction between P10 and MYC-Promoter (MYC-Pro) and temporal epigenetic repression of these MYC-proximal elements. We next designed a CRISPR/Cas9-mediated double genomic knock-out (KO) strategy to show that P10-KO slightly lessened androgen-elicited MYC transrepression in LNCaP-AR cells. In similar genomic editing assays, androgen-mediated MYC repression became slightly deepened upon KO of P11, an ABS in the PVT1 gene locus highly enriched in AR-binding motifs and peaks. We also investigated multiple ABSs in the established PCAT1 super-enhancer that distally interacts with MYC-Pro for transactivation, with each KO pool consistently shown to relieve androgen-elicited MYC repression. In the end, we systemically assessed androgen effects in the 8q24-MYC locus and along PCa genome to generalize H3K27ac and BRD4 re-distribution from pioneer factors (FoxA1 and HoxB13) to AR sites. CONCLUSION: Together, we reconciled these observations by unifying AR dual-functions that are mechanistically coupled to and equilibrated by co-factor redistribution.

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis.

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic "HAP" pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically "normal" capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

Recent advances in anticancer peptoids.

Since most tumors become resistant to drugs in a gradual and irreversible manner, making treatment less effective over time, anticancer drugs require continuous development. Peptoids are a class of peptidomimetics that can be easily synthesized and optimized. They exhibit a number of unique characteristics, including protease resistance, non-immunogenicity, do not interfere with peptide functionality and skeleton polarity, and can adopt different conformations. They have been studied for their efficacy in different cancer therapies, and can be considered as a promising alternative molecular category for the development of anticancer drugs. Herein, we discuss the extensive recent advances in peptoids and peptoid hybrids in the treatment of cancers such as prostate, breast, lung, and other ones, in the hope of providing a reference for the further development of peptoid anticancer drugs.

High expression of ABCF1 is an independent predictor of poor prognosis in bladder cancer.

ABCF1, a member of the ATP-binding cassette (ABC) transporter family, is involved in the malignant progression of tumors. However, the role of ABCF1 in bladder cancer is poorly understood. In our study, we explored the differential expression of ABCF1 in bladder cancer and normal bladder tissues based on bioinformatic analysis and immunohistochemical results. GSEA was performed to ascertain the potential related signaling pathways of ABCF1. The relationship between ABCF1 expression and bladder cancer progression was analyzed using the GSE13507 dataset. In addition, the differential expression of ABCF1 in the cell lines was verified by quantitative real-time polymerase chain reaction (qRT‒PCR) and Western blotting. ABCF1 was upregulated in bladder cancer, and the high expression of ABCF1 was closely related to sex (P = 0.00056), grade (P = 0.00049), T stage (P = 0.00007), and N stage (P = 0.0076). High expression of ABCF1 was correlated with poor overall survival in bladder cancer patients (P < 0.001). In addition, univariate and multivariate Cox regression analyses showed that high ABCF1 expression was an independent factor for poor prognosis in bladder cancer patients. Therefore, ABCF1 expression is closely related to the progression of bladder cancer and can be used as a potential indicator of poor prognosis and a therapeutic target for bladder cancer.

Progress in the Total Synthesis of Antitumor Tetrahydroisoquinoline Alkaloids.

Among the tetrahydroisoquinoline(THIQ) of natural products, a family of THIQ alkaloids has the characteristics of similar biosynthetic pathway. Such THIQ alkaloids family mainly include Renieramycins, Ecteinasicdins, Tetrazaomine, Lemonomycin, etc. Most of these natural compounds have strong antitumor activities, and its family member Ecteinasicdins743 (ET-743, Trabectedin) has been marketed in the European Union and the United States for the treatment of advanced soft tissue tumors and ovarian cancer. Because of the excellent biological activity and complex chemical structure of this kind of THIQ products, it has aroused great interest of biologists and chemists, and many synthetic chemists have paid considerable efforts to their total synthesis over the past decade. Based on this, the recent advances in the total synthesis of such THIQ alkaloids are reviewed.

Design, synthesis, and biological evaluation of simplified tetrahydroisoquinoline analogs.

A series of tetrahydroisoquinoline derivatives were prepared and their antitumor activity was studied against several human carcinoma cell lines, including Ketr3, BEL-7402, BGC-823, KB, HCT-8, MCF-7, HeLa, A2780, A549, and HT-1080. Compound 20, an analog of phthalascidin 650, exhibited good broad-spectrum antitumor activity in vitro. However, compounds 19 and 21, in which the side chains at C-22 are simplified, showed no obvious antitumor activity, indicating that the C-22 side chain of this type of compound has a greater impact on its activity. The difference in the in vivo activity between compound 20 and phthalascidin 650 also shows a significant effect of the substituents on the skeleton structure on the in vivo activity.

A putative amphipathic alpha helix in hepatitis B virus small envelope protein plays a critical role in the morphogenesis of subviral particles.

Hepatitis B virus (HBV) small (S) envelope protein has the intrinsic ability to direct the formation of small spherical subviral particles (SVPs) in eukaryotic cells. However, the molecular mechanism underlying the morphogenesis of SVPs from the monomeric S protein initially synthesized at the endoplasmic reticulum (ER) membrane remains largely elusive. Structure prediction and extensive mutagenesis analysis suggested that the amino acid residues spanning W156 to R169 of S protein form an amphipathic alpha helix and play essential roles in SVP production and S protein metabolic stability. Further biochemical analyses showed that the putative amphipathic alpha helix was not required for the disulfide-linked S protein oligomerization, but was essential for SVP morphogenesis. Pharmacological disruption of vesicle trafficking between the ER and Golgi complex in SVP producing cells supported the hypothesis that S protein-directed SVP morphogenesis takes place at the ER-Golgi intermediate compartment (ERGIC). Moreover, it was demonstrated that S protein is degraded in hepatocytes via a 20S proteasome-dependent, but ubiquitination-independent non-classic ER-associated degradation (ERAD) pathway. Taken together, the results reported herein favor a model in which the amphipathic alpha helix at the antigenic loop of S protein attaches to the lumen leaflet to facilitate SVP budding from the ERGIC compartment, whereas the failure of budding process may result in S protein degradation by 20S proteasome in an ubiquitination-independent manner.Importance Subviral particles are the predominant viral product produced by HBV-infected hepatocytes. Their levels exceed the virion particles by 10,000 to 100,000-fold in the blood of HBV infected individuals. The high levels of SVPs, or HBV surface antigen (HBsAg), in the circulation induces immune tolerance and contributes to the establishment of persistent HBV infection. The loss of HBsAg, often accompanied by appearance of anti-HBs antibodies, is the hallmark of durable immune control of HBV infection. Therapeutic induction of HBsAg loss is, therefore, considered to be essential for the restoration of host antiviral immune response and functional cure of chronic hepatitis B. Our findings on the mechanism of SVP morphogenesis and S protein metabolism will facilitate the rational discovery and development of antiviral drugs to achieve this therapeutic goal.

Protein phosphatase 1 catalyzes HBV core protein dephosphorylation and is co-packaged with viral pregenomic RNA into nucleocapsids.

Hepatitis B virus (HBV) replicates its genomic DNA via viral DNA polymerase self-primed reverse transcription of a RNA pre-genome in the nucleocapsid assembled by 120 core protein (Cp) dimers. The arginine-rich carboxyl-terminal domain (CTD) of Cp plays an important role in the selective packaging of viral DNA polymerase-pregenomic (pg) RNA complex into nucleocapsid. Previous studies suggested that the CTD is initially phosphorylated at multiple sites to facilitate viral RNA packaging and subsequently dephosphorylated in association with viral DNA synthesis and secretion of DNA-containing virions. However, our recent studies suggested that Cp is hyper-phosphorylated as free dimers and its dephosphorylation is associated with pgRNA encapsidation. Herein, we provide further genetic and biochemical evidence supporting that extensive Cp dephosphorylation does take place during the assembly of pgRNA-containing nucleocapsids, but not empty capsids. Moreover, we found that cellular protein phosphatase 1 (PP1) is required for Cp dephosphorylation and pgRNA packaging. Interestingly, the PP1 catalytic subunits α and ß were packaged into pgRNA-containing nucleocapsids, but not empty capsids, and treatment of HBV replicating cells with core protein allosteric modulators (CpAMs) promoted empty capsid assembly and abrogated the encapsidation of PP1 α and ß. Our study thus identified PP1 as a host cellular factor that is co-packaged into HBV nucleocapsids, and plays an essential role in selective packaging of the viral DNA-polymerase-pgRNA complex through catalyzing Cp dephosphorylation.

4-Oxooctahydroquinoline-1(2H)-carboxamides as hepatitis B virus (HBV) capsid core protein assembly modulators.

Hepatitis B virus (HBV) core protein, the building block of the HBV capsid, plays multiple roles in viral replication, and is an attractive target for development of antiviral agents with a new mechanism of action. In addition to the heteroaryldihydropyrimidines (HAPs), sulfamoylbenzamides (SBAs), dibenzothiazepine derivatives (DBTs), and sulfamoylpyrrolamides (SPAs) that inhibit HBV replication by modulation of viral capsid assembly and are currently under clinical trials for the treatment of chronic hepatitis B (CHB), other chemical structures with activity to modulate HBV capsid assembly have also been explored. Here we describe our continued optimization of a benzamide originating from our high throughput screening. A new bicyclic carboxamide lead featuring an electron deficient non-planar core structure was discovered. Evaluations of its ADMET (absorption, distribution, metabolism, excretion and toxicity) and pharmacokinetic (PK) profiles demonstrate improved metabolic stability and good bioavailability.

A new biomarker to enhance the radiosensitivity of hepatocellular cancer: miRNAs.

Aims: This review summarizes findings regarding miRNAs that modulate radiation in hepatocellular carcinoma (HCC) and evaluates their potential clinical therapeutic uses. Materials & methods: We searched the relevant English-language medical databases for papers on miRNAs and radiation therapy for tumors to identify miRNAs that are linked with radiosensitivity and radioresistance, focusing on those associated with HCC radiation. Results: There were 88 papers assessed for miRNAs associated with tumor radiation, 56 of which dealt with radiosensitization, 21 with radioresistance and 11 with radiosensitization for HCC. Conclusion: Further work in this area would enable future evaluation of radiation responses and the potential use of miRNAs as therapeutic agents in HCC patients.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, with high mortality and poor clinical outcomes. Radiotherapy is necessary for around 70% of cancer patients. As the role of miRNAs in regulating tumor radiosensitivity is more investigated, their significance in the development of HCC and their potential to alter the function of radiation in HCC become increasingly apparent. This review addresses the function of miRNAs in controlling radiation in cancer cells, concentrating on miRNA expression during radiosensitization of HCC and therapeutic uses of these results.