Mechanism of interferon alpha therapy for chronic hepatitis B and potential approaches to improve its therapeutic efficacy.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide and causes more than 820,000 deaths annually due to cirrhosis and hepatocellular carcinoma. Current standard-of-care medications for chronic hepatitis B (CHB) include nucleos(t)ide analogue (NA) viral DNA polymerase inhibitors and pegylated interferon alpha (PEG-IFN-α). NAs can efficiently suppress viral replication and improve liver pathology, but not eliminate or inactivate HBV covalently closed circular DNA (cccDNA). CCC DNA is the most stable HBV replication intermediate that exists as a minichromosome in the nucleus of infected hepatocyte to transcribe viral RNA and support viral protein translation and genome replication. Consequentially, a finite duration of NA therapy rarely achieves a sustained off-treatment suppression of viral replication and life-long NA treatment is most likely required. On the contrary, PEG-IFN-α has the benefit of finite treatment duration and achieves HBsAg seroclearance, the indication of durable immune control of HBV replication and functional cure of CHB, in approximately 5% of treated patients. However, the low antiviral efficacy and poor tolerability limit its use. Understanding how IFN-α suppresses HBV replication and regulates antiviral immune responses will help rational optimization of IFN therapy and development of novel immune modulators to improve the rate of functional cure. This review article highlights mechanistic insight on IFN control of HBV infection and recent progress in development of novel IFN regimens, small molecule IFN mimetics and combination therapy of PEG-IFN-α with new direct-acting antivirals and therapeutic vaccines to facilitate the functional cure of CHB.

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.

RNA binding protein TIAR modulates HBV replication by tipping the balance of pgRNA translation.

The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves not only as a bicistronic message RNA to translate core protein (Cp) and DNA polymerase (Pol), but also as the template for reverse transcriptional replication of viral DNA upon packaging into nucleocapsid. Although it is well known that pgRNA translates much more Cp than Pol, the molecular mechanism underlying the regulation of Cp and Pol translation efficiency from pgRNA remains elusive. In this study, we systematically profiled HBV nucleocapsid- and pgRNA-associated cellular proteins by proteomic analysis and identified TIA-1-related protein (TIAR) as a novel cellular protein that binds pgRNA and promotes HBV DNA replication. Interestingly, loss- and gain-of-function genetic analyses showed that manipulation of TIAR expression did not alter the levels of HBV transcripts nor the secretion of HBsAg and HBeAg in human hepatoma cells supporting HBV replication. However, Ribo-seq and PRM-based mass spectrometry analyses demonstrated that TIAR increased the translation of Pol but decreased the translation of Cp from pgRNA. RNA immunoprecipitation (RIP) and pulldown assays further revealed that TIAR directly binds pgRNA at the 5' stem-loop (ε). Moreover, HBV replication or Cp expression induced the increased expression and redistribution of TIAR from the nucleus to the cytoplasm of hepatocytes. Our results thus imply that TIAR is a novel cellular factor that regulates HBV replication by binding to the 5' ε structure of pgRNA to tip the balance of Cp and Pol translation. Through induction of TIAR translocation from the nucleus to the cytoplasm, Cp indirectly regulates the Pol translation and balances Cp and Pol expression levels in infected hepatocytes to ensure efficient viral replication.

Identification of novel tetrahydroquinoxaline derived phenyl ureas as modulators of the hepatitis B virus nucleocapsid assembly.

A key step of hepatitis B virus (HBV) replication is the selective packaging of pregenomic RNA (pgRNA) by core protein (Cp) dimers, forming a nucleocapsid where the reverse transcriptional viral DNA replication takes place. One approach in the development of new anti-HBV drugs is to disrupt the assembly of HBV nucleocapsids by misdirecting Cp dimers to assemble morphologically normal capsids devoid of pgRNA. In this study, we built upon our previous discovery of benzamide-derived HBV capsid assembly modulators by exploring fused bicyclic scaffolds with an exocyclic amide that is ß, γ to the fused ring, and identified 1,2,3,4-tetrahydroquinoxaline derived phenyl ureas as a novel scaffold. Structure-activity relationship studies showed that a favorable hydrophobic substitution can be tolerated at the 2-position of the 1,2,3,4-tetrahydroquinoxaline core, and the resulting compound 88 demonstrated comparable or improved antiviral potencies in mouse and human hepatocyte-derived HBV-replicating cell lines compared to our previously reported benzamide compound, 38017 (8). In addition, a novel bis-urea series based on 1,2,3,4-tetrahydroquinoxaline was also found to inhibit HBV DNA replication with sub-micromolar EC50 values. The mode of action of these compounds is consistent with specific inhibition of pgRNA encapsidation into nucleocapsids in hepatocytes.

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.

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.

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.

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.

Interferon Control of Human Coronavirus Infection and Viral Evasion: Mechanistic Insights and Implications for Antiviral Drug and Vaccine Development.

Recognition of viral infections by various pattern recognition receptors (PRRs) activates an inflammatory cytokine response that inhibits viral replication and orchestrates the activation of adaptive immune responses to control the viral infection. The broadly active innate immune response puts a strong selective pressure on viruses and drives the selection of variants with increased capabilities to subvert the induction and function of antiviral cytokines. This revolutionary process dynamically shapes the host ranges, cell tropism and pathogenesis of viruses. Recent studies on the innate immune responses to the infection of human coronaviruses (HCoV), particularly SARS-CoV-2, revealed that HCoV infections can be sensed by endosomal toll-like receptors and/or cytoplasmic RIG-I-like receptors in various cell types. However, the profiles of inflammatory cytokines and transcriptome response induced by a specific HCoV are usually cell type specific and determined by the virus-specific mechanisms of subverting the induction and function of interferons and inflammatory cytokines as well as the genetic trait of the host genes of innate immune pathways. We review herein the recent literatures on the innate immune responses and their roles in the pathogenesis of HCoV infections with emphasis on the pathobiological roles and therapeutic effects of type I interferons in HCoV infections and their antiviral mechanisms. The knowledge on the mechanism of innate immune control of HCoV infections and viral evasions should facilitate the development of therapeutics for induction of immune resolution of HCoV infections and vaccines for efficient control of COVID-19 pandemics and other HCoV infections.

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.

Prospects for the Global Elimination of Hepatitis B.

Chronic hepatitis B virus (HBV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma, estimated to be globally responsible for ∼800,000 deaths annually. Although effective vaccines are available to prevent new HBV infection, treatment of existing chronic hepatitis B (CHB) is limited, as the current standard-of-care antiviral drugs can only suppress viral replication without achieving cure. In 2016, the World Health Organization called for the elimination of viral hepatitis as a global public health threat by 2030. The United States and other nations are working to meet this ambitious goal by developing strategies to cure CHB, as well as prevent HBV transmission. This review considers recent research progress in understanding HBV pathobiology and development of therapeutics for the cure of CHB, which is necessary for elimination of hepatitis B by 2030.

Restoration of a functional antiviral immune response to chronic HBV infection by reducing viral antigen load: if not sufficient, is it necessary?

The prolonged viral antigen stimulation is the driving force for the development of immune tolerance to chronic hepatitis B virus (HBV) infection. The sustained reduction of viral proteins may allow for the recovery and efficient activation of HBV-specific T and B cells by immune-stimulating agents, checkpoint blockades and/or therapeutic vaccinations. Recently, several therapeutic approaches have been shown to significantly reduce intrahepatic viral proteins and/or circulating HBV surface antigen (HBsAg) with variable impacts on the host antiviral immune responses in animal models or human clinical trials. It remains to be further investigated whether reduction of viral protein expression or induction of intrahepatic viral protein degradation is more efficacious to break the immune tolerance to chronic HBV infection. It is also of great interest to know if the accelerated clearance of circulating HBsAg by antibodies has a long-term immunological impact on HBV infection and disease progression. Although it is clear that removal of antigen stimulation alone is not sufficient to induce the functional recovery of exhausted T and B cells, accumulating evidence suggests that the reduction of viral antigen load appears to facilitate the therapeutic activation of functional antiviral immunity in chronic HBV carriers. Based on a systematic review of the findings in animal models and clinical studies, the research directions toward discovery and development of more efficacious therapeutic approaches to reinvigorate HBV-specific adaptive immune function and achieve the durable control of chronic HBV infection, i.e. a functional cure, in the vast majority of treated patients are discussed.

Identification of hepatitis B virus core protein residues critical for capsid assembly, pgRNA encapsidation and resistance to capsid assembly modulators.

Assembly of hepatitis B virus (HBV) capsids is driven by the hydrophobic interaction of core protein (Cp) at dimer-dimer interface. Binding of core protein allosteric modulators (CpAMs) to a hydrophobic "HAP" pocket formed between the inter-dimer interface strengths the dimer-dimer interaction and misdirects the assembly of Cp dimers into non-capsid Cp polymers or morphologically normal capsids devoid of viral pregenomic (pg) RNA and DNA polymerase. In this study, we performed a systematic mutagenesis analysis to identify Cp amino acid residues at Cp dimer-dimer interface that are critical for capsid assembly, pgRNA encapsidation and resistance to CpAMs. By analyzing 70 mutant Cp with a single amino acid substitution of 25 amino acid residues around the HAP pocket, our study revealed that residue W102 and Y132 are critical for capsid assembly. However, substitution of many other residues did not significantly alter the amount of capsids, but reduced the amount of encapsidated pgRNA, suggesting their critical roles in pgRNA packaging. Interestingly, several mutant Cp with a single amino acid substitution of residue P25, T33 or I105 supported high levels of DNA replication, but conferred strong resistance to multiple chemotypes of CpAMs. In addition, we also found that WT Cp, but not the assembly incompetent Cp, such as Y132A Cp, interacted with HBV DNA polymerase (Pol). This later finding implies that encapsidation of viral DNA polymerase may depend on the interaction of Pol with a capsid assembly intermediate, but not free Cp dimers. Taking together, our findings reported herein shed new light on the mechanism of HBV nucleocapsid assembly and mode of CpAM action.

Hepatitis B virus nucleocapsid uncoating: biological consequences and regulation by cellular nucleases.

Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both de novo infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from de novo HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5' end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during de novo infection may occur at a different subcellular compartment and possibly via distinct mechanisms.

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.

Synthesis of 4-oxotetrahydropyrimidine-1(2H)-carboxamides derivatives as capsid assembly modulators of hepatitis B virus.

We report herein the synthesis and evaluation of phenyl ureas derived from 4-oxotetrahydropyrimidine as novel capsid assembly modulators of hepatitis B virus (HBV). Among the derivatives, compound 27 (58031) and several analogs showed an activity of submicromolar EC50 against HBV and low cytotoxicities (>50 µM). Structure-activity relationship studies revealed a tolerance for an additional group at position 5 of 4-oxotetrahydropyrimidine. The mechanism study indicates that compound 27 (58031) is a type II core protein allosteric modulator (CpAMs), which induces core protein dimers to assemble empty capsids with fast electrophoresis mobility in native agarose gel. These compounds may thus serve as leads for future developments of novel antivirals against HBV.

Targeting the multifunctional HBV core protein as a potential cure for chronic hepatitis B.

The core (capsid) protein of hepatitis B virus (HBV) is the building block of nucleocapsids where viral DNA reverse transcriptional replication takes place and mediates virus-host cell interaction important for the persistence of HBV infection. The pleiotropic role of core protein (Cp) in HBV replication makes it an attractive target for antiviral therapies of chronic hepatitis B, a disease that affects more than 257 million people worldwide without a cure. Recent clinical studies indicate that core protein allosteric modulators (CpAMs) have a great promise as a key component of hepatitis B curative therapies. Particularly, it has been demonstrated that modulation of Cp dimer-dimer interactions by several chemical series of CpAMs not only inhibit nucleocapsid assembly and viral DNA replication, but also induce the disassembly of double-stranded DNA-containing nucleocapsids to prevent the synthesis of cccDNA. Moreover, the different chemotypes of CpAMs modulate Cp assembly by interaction with distinct amino acid residues at the HAP pocket between Cp dimer-dimer interfaces, which results in the assembly of Cp dimers into either non-capsid Cp polymers (type I CpAMs) or empty capsids with distinct physical property (type II CpAMs). The different CpAMs also differentially modulate Cp metabolism and subcellular distribution, which may impact cccDNA metabolism and host antiviral immune responses, the critical factors for the cure of chronic HBV infection. This review article highlights the recent research progress on the structure and function of core protein in HBV replication cycle, the mode of action of CpAMs, as well as the current status and perspectives on the discovery and development of core protein-targeting antivirals. This article forms part of a symposium in Antiviral Research on "Wide-ranging immune and direct-acting antiviral approaches to curing HBV and HDV infections."

Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus.

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.

Bat SARS-Like WIV1 coronavirus uses the ACE2 of multiple animal species as receptor and evades IFITM3 restriction via TMPRSS2 activation of membrane fusion.

Diverse SARS-like coronaviruses (SL-CoVs) have been identified from bats and other animal species. Like SARS-CoV, some bat SL-CoVs, such as WIV1, also use angiotensin converting enzyme 2 (ACE2) from human and bat as entry receptor. However, whether these viruses can also use the ACE2 of other animal species as their receptor remains to be determined. We report herein that WIV1 has a broader tropism to ACE2 orthologs than SARS-CoV isolate Tor2. Among the 9 ACE2 orthologs examined, human ACE2 exhibited the highest efficiency to mediate the infection of WIV1 pseudotyped virus. Our findings thus imply that WIV1 has the potential to infect a wide range of wild animals and may directly jump to humans. We also showed that cell entry of WIV1 could be restricted by interferon-induced transmembrane proteins (IFITMs). However, WIV1 could exploit the airway protease TMPRSS2 to partially evade the IFITM3 restriction. Interestingly, we also found that amphotericin B could enhance the infectious entry of SARS-CoVs and SL-CoVs by evading IFITM3-mediated restriction. Collectively, our findings further underscore the risk of exposure to animal SL-CoVs and highlight the vulnerability of patients who take amphotericin B to infection by SL-CoVs, including the most recently emerging (SARS-CoV-2).

LY6E Restricts Entry of Human Coronaviruses, Including Currently Pandemic SARS-CoV-2.

C3A is a subclone of the human hepatoblastoma HepG2 cell line with strong contact inhibition of growth. We fortuitously found that C3A was more susceptible to human coronavirus HCoV-OC43 infection than HepG2, which was attributed to the increased efficiency of virus entry into C3A cells. In an effort to search for the host cellular protein(s) mediating the differential susceptibility of the two cell lines to HCoV-OC43 infection, we found that ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and lymphocyte antigen 6 family member E (LY6E), the three cellular proteins identified to function in interference with virus entry, were expressed at significantly higher levels in HepG2 cells. Functional analyses revealed that ectopic expression of LY6E, but not GILT or ADAP2, in HEK 293 cells inhibited the entry of HCoV-O43. While overexpression of LY6E in C3A and A549 cells efficiently inhibited the infection of HCoV-OC43, knockdown of LY6E expression in HepG2 significantly increased its susceptibility to HCoV-OC43 infection. Moreover, we found that LY6E also efficiently restricted the entry mediated by the envelope spike proteins of other human coronaviruses, including the currently pandemic SARS-CoV-2. Interestingly, overexpression of serine protease TMPRSS2 or amphotericin treatment significantly neutralized the IFN-inducible transmembrane 3 (IFITM3) restriction of human coronavirus (CoV) entry, but did not compromise the effect of LY6E on the entry of human coronaviruses. The work reported herein thus demonstrates that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis via a mechanism distinct from other factors that modulate CoV entry.IMPORTANCE Virus entry into host cells is one of the key determinants of host range and cell tropism and is subjected to the control of host innate and adaptive immune responses. In the last decade, several interferon-inducible cellular proteins, including IFITMs, GILT, ADAP2, 25CH, and LY6E, had been identified to modulate the infectious entry of a variety of viruses. Particularly, LY6E was recently identified as a host factor that facilitates the entry of several human-pathogenic viruses, including human immunodeficiency virus, influenza A virus, and yellow fever virus. Identification of LY6E as a potent restriction factor of coronaviruses expands the biological function of LY6E and sheds new light on the immunopathogenesis of human coronavirus infection.