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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. Author summary: Emergence and re-emergence of yellow fever (YF) caused by the yellow fever virus (YFV) infection have posed a global public health threat in previously non-epidemic as well as endemic regions. The approximately 30% of mortality rate makes the outbreaks particularly devastating. In addition to the vaccination campaign and mosquito controls, antiviral drugs are important components in the toolbox for combating YF outbreaks. However, only two nucleotide analogue drugs developed for the treatment of other RNA virus infections are currently repurposed for the treatment of YF with uncertain clinical efficacy. BDAA is a benzodiazepine compound discovered as a potent YFV-specific antiviral agent in our laboratory. The work reported herein further demonstrates that BDAA interaction with the YFV NS4B protein may impair the integrity of viral RNA replication organelles, which not only inhibits viral RNA replication, but also results in the leakage of viral RNA into the cytoplasm to activate RIG-I-like RNA receptors and enhances the innate antiviral immune response. The unprecedented antiviral mechanism of BDAA highlights the essential role of the NS4B protein in viral RNA replication and the evasion of host cellular innate immunity. [ABSTRACT FROM AUTHOR]

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. Author summary: Binding of CpAMs at a HAP pocket between Cp dimer-dimer interfaces misdirects HBV capsid assembly. We demonstrated herein that CpAM-resistant mutations with single amino acid substitution of Cp residues at the HAP pocket not only confer resistance to CpAM suppression of pgRNA packaging, but also to the induction of capsid structure alteration and mature nucleocapsid uncoating as well as the inhibition of cccDNA synthesis by GLS4, a type I CpAM currently in phase 2 clinical trials. Moreover, these mutations in the context of precore protein also conferred resistance to CpAM inhibition of HBeAg secretion. We have thus obtained genetic evidence suggesting that CpAMs disrupt the multiple steps of HBV replication and inhibit HBeAg secretion by misdirecting the interaction of Cp or reduced p17 dimers at the HAP pocket. The mechanistic insights gained from this study should facilitate the development of Cp/p17-targeting antiviral drugs for the treatment of chronic hepatitis B. [ABSTRACT FROM AUTHOR]

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 β 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 β. 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. Author summary: Selective packaging of pregenomic RNA by core protein dimers into nucleocapsid is a key step of HBV replication and is subjected for the regulation by multiple viral and host cellular factors. HBV core protein phosphorylation and dephosphorylation play an essential role in HBV genome replication. However, the cellular kinases and phosphatases responsible for the biochemical events remain elusive. Identification of cellular protein phosphatase 1 as a host cellular factor catalyzing core protein dephosphorylation and facilitating viral pregenomic RNA packaging into nucleocapsids sheds new light on the molecular mechanism of HBV replication and development of therapeutics to cure chronic HBV infection. [ABSTRACT FROM AUTHOR]

Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol κ and Pol λ. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. [ABSTRACT FROM AUTHOR]

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/βR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen. [ABSTRACT FROM AUTHOR]

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection. [ABSTRACT FROM AUTHOR]

Tripartite motif (TRIM) proteins constitute a family of over 100 members that share conserved tripartite motifs and exhibit diverse biological functions. Several TRIM proteins have been shown to restrict viral infections and regulate host cellular innate immune responses. In order to identify TRIM proteins that modulate the infection of hepatitis B virus (HBV), we tested 38 human TRIMs for their effects on HBV gene expression, capsid assembly and DNA synthesis in human hepatoma cells (HepG2). The study revealed that ectopic expression of 8 TRIM proteins in HepG2 cells potently reduced the amounts of secreted HBV surface and e antigens as well as intracellular capsid and capsid DNA. Mechanistic analyses further demonstrated that the 8 TRIMs not only reduced the expression of HBV mRNAs, but also inhibited HBV enhancer I and enhancer II activities. Studies focused on TRIM41 revealed that a HBV DNA segment spanning nucleotide 1638 to nucleotide 1763 was essential for TRIM41-mediated inhibition of HBV enhancer II activity and the inhibitory effect depended on the E3 ubiquitin ligase activity of TRIM41 as well as the integrity of TRIM41 C-terminal domain. Moreover, knockdown of endogenous TRIM41 in a HepG2-derived stable cell line significantly increased the level of HBV preC/C RNA, leading to an increase in viral core protein, capsid and capsid DNA. Our studies have thus identified eight TRIM proteins that are able to inhibit HBV transcription and provided strong evidences suggesting the endogenous role of TRIM41 in regulating HBV transcription in human hepatoma cells. [ABSTRACT FROM AUTHOR]

The zinc finger antiviral protein (ZAP) is a mammalian host restriction factor that inhibits the replication of a variety of RNA viruses, including retroviruses, alphaviruses and filoviruses, through interaction with the ZAP-responsive elements (ZRE) in viral RNA, and recruiting the exosome to degrade RNA substrate. Hepatitis B virus (HBV) is a pararetrovirus that replicates its genomic DNA via reverse transcription of a viral pregenomic (pg) RNA precursor. Here, we demonstrate that the two isoforms of human ZAP (hZAP-L and -S) inhibit HBV replication in human hepatocyte-derived cells through posttranscriptional down-regulation of viral pgRNA. Mechanistically, the zinc finger motif-containing N-terminus of hZAP is responsible for the reduction of HBV RNA, and the integrity of the four zinc finger motifs is essential for ZAP to bind to HBV RNA and fulfill its antiviral function. The ZRE sequences conferring the susceptibility of viral RNA to ZAP-mediated RNA decay were mapped to the terminal redundant region (nt 1820–1918) of HBV pgRNA. In agreement with its role as a host restriction factor and as an innate immune mediator for HBV infection, ZAP was upregulated in cultured primary human hepatocytes and hepatocyte-derived cells upon IFN-α treatment or IPS-1 activation, and in the livers of hepatitis B patients during immune active phase. Knock down of ZAP expression increased the level of HBV RNA and partially attenuated the antiviral effect elicited by IPS-1 in cell cultures. In summary, we demonstrated that ZAP is an intrinsic host antiviral factor with activity against HBV through down-regulation of viral RNA, and that ZAP plays a role in the innate control of HBV replication. Our findings thus shed light on virus-host interaction, viral pathogenesis, and antiviral approaches. [ABSTRACT FROM AUTHOR]

Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.

Recognition of virus infection by innate pattern recognition receptors (PRRs), including membrane-associated toll-like receptors (TLR) and cytoplasmic RIG-I-like receptors (RLR), activates cascades of signal transduction pathways leading to production of type I interferons (IFN) and proinflammatory cytokines that orchestrate the elimination of the viruses. Although it has been demonstrated that PRR-mediated innate immunity plays an essential role in defending virus from infection, it also occasionally results in overwhelming production of proinflammatory cytokines that cause severe inflammation, blood vessel leakage and tissue damage. In our efforts to identify small molecules that selectively enhance PRR-mediated antiviral, but not the detrimental inflammatory response, we discovered a compound, RO 90-7501 ('2'-(4-Aminophenyl)-[2,5'-bi-1H-benzimidazol]-5-amine), that significantly promoted both TLR3 and RLR ligand-induced IFN-β gene expression and antiviral response, most likely via selective activation of p38 mitogen-activated protein kinase (MAPK) pathway. Our results thus imply that pharmacological modulation of PRR signal transduction pathways in favor of the induction of a beneficial antiviral response can be a novel therapeutic strategy.