Virus Core Research Articles

Discovery of SHR5133, a Highly Potent and Novel HBV Capsid Assembly Modulator.

Capsid assembly modulators (CpAMs) represent a new class of antivirals targeting hepatitis B virus (HBV) core protein to disrupt the assembly process. In this work, a novel chemotype featuring a fused heterocycle amide was discovered through pharmacophore exploration. Lead optimization resulted in compound 8 with an EC50 value of 511 nM, and then methyl substitution on the piperazine was found to improve the in vitro potency remarkably. Further SAR studies established the key compound SHR5133, which showed high in vitro antiviral potency, favorable pharmacokinetic profiles across species, and robust in vivo efficacy.

TRIM5α Restriction of HIV-1-N74D Viruses in Lymphocytes Is Caused by a Loss of Cyclophilin A Protection.

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein CPSF6. In primary human CD4+ T cells, HIV-1-N74D viruses exhibit an infectivity defect when compared to wild-type. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4+ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D viruses is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5αhu) restriction in CD4+ T cells, we found that depletion of TRIM5αhu in CD4+ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5αhu. Accordingly, TRIM5αhu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In agreement with the notion that N74D capsids are defective in their ability to bind Cyp A, we found that HIV-1-N74D viruses were 20-fold less sensitive to TRIMCyp restriction when compared to wild-type viruses in OMK cells. Structural analysis revealed that N74D hexameric capsid protein in complex with PF74 is different from wild-type hexameric capsid protein in complex with PF74, which explains the defect of N74D capsids to interact with Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4+ T cells is due to a loss of Cyp A protection from TRIM5αhu restriction activity.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a 'pull-down assay' used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a 'pull-down assay' used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

N6-methyladenosine modification of the 5′ epsilon structure of the HBV pregenome RNA regulates its encapsidation by the viral core protein

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5' end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m6A)-modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m6A modification of the 5' ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m6A site mutant in the HBV 5' epsilon, we present evidence that m6A methylation of 5' epsilon is necessary for its encapsidation. The m6A modification of 5' epsilon increased the efficiency of viral RNA packaging, whereas the m6A of 3' epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m6A modification at 5' epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5' epsilon m6A site-mutated pgRNA failed to interact. HBV polymerase interaction with 5' epsilon was independent of m6A modification of 5' epsilon. This study highlights yet another pivotal role of m6A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA-protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m6A modification.

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

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

Virus breathing, metastability and epitope dynamics

RNA viruses are metastable macromolecular assemblies that sense their environment through dynamic breathing motions. Amide hydrogen/exchange mass spectrometry (HDXMS) is a powerful method to uncover dynamics of whole viral particles in solution. I will describe dynamics of viral surface regions and core ribonucleoprotein in two model viruses: Dengue and Turnip Crinkle Virus (TCV). We have identified a broad neutralizing antibody, C10 that induces altered ‘breathing’ dynamics in dengue viral particles and shows unique stoichiometry-specific responses.

Single-molecule binding studies of antiviral compounds targeting the hepatitis c virus core protein

Of the 10 different proteins associated with the hepatitis C virus (HCV), the HCV core protein (HCVcp) is the most conserved. HCVcp is a nucleocapsid protein that plays a crucial role in viral assembly and therefore it is a promising target for antiviral therapeutics. However, the intrinsically disorder nature of the protein leads to a high-degree of conformational heterogeneity, which makes it challenging to study biochemical interactions between HCVcp and potential therapeutics using classical structure-based approaches.

Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection.

New insights into the role of endosomal proteins for African swine fever virus infection.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.

Persistence of Immunity in Adults after 1, 5 and 10 Years with Recombinant Hepatitis B Vaccine in Beijing in 2010-2020.

The persistence of immunity after hepatitis B vaccination is still under investigation in adults. In Chaoyang District, Beijing, people who were aged ≥ 18 years and completely immunized with HBV vaccine according to the standard procedure (0–1–6 months) were enrolled. Three groups were set for 1 (Y1), 5 (Y5) and 10 (Y10) years after the hepatitis B vaccination. The following data was collected and analyzed: antibody against hepatitis B virus surface antigen(anti-HBs) positive rates and geometric mean concentration (GMC) between the different compared groups through questionnaires and laboratory detection, including hepatitis B virus surface antigen (HBsAg), anti-HBs and antibody against hepatitis B virus core antigen(anti-HBc). All 600 subjects completed the questionnaires and serological tests. Among all subjects, the positive rates of HBsAg, anti-HBs and anti-HBc were 0, 70.5% (423/600) and 2.5% (15/600), respectively. The anti-HBs positive rates in Y1, Y5 and Y10 groups were 86.5% (173/200), 71.0% (142/200) and 54.0% (108/200) (χ2 = 50.8, p < 0.001) and showed a linear decreasing trend year by year (trend χ2 = 50.7, p < 0.001). The GMC in Y1, Y5 and Y10 groups were 296.6 mIU/mL, 51.6 mIU/mL and 25.5 mIU/mL (H = 64.8, p < 0.001), respectively. The anti-HBs positive rates and GMC decreased rapidly after the vaccination of adults against hepatitis B. Screening after 5–10 years and booster vaccination for the unprotected population is recommended.

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein–Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

Since the beginning of the COVID-19 pandemic, the development of effective vaccines against this pathogen has been a priority for the scientific community. Several strategies have been developed including vaccines based on recombinant viral protein fragments. The receptor-binding domain (RBD) in the S1 subunit of S protein has been considered one of the main targets of neutralizing antibodies. In this study we assess the potential of a vaccine formulation based on the recombinant RBD domain of SARS-CoV-2 expressed in the thermophilic filamentous fungal strain Thermothelomyces heterothallica and the hepatitis B virus (HBV) core protein. Functional humoral and cellular immune responses were detected in mice. To our knowledge, this is the first report on the immune evaluation of a biomedical product obtained in the fungal strain T. heterothallica. These results together with the intrinsic advantages of this expression platform support its use for the development of biotechnology products for medical purpose.

All-trans retinoic acid inhibits HCV replication by downregulating core levels via E6AP-mediated proteasomal degradation

Here, we found that all-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, strengthens the anti-viral defense mechanism of E6-associated protein (E6AP) that downregulates hepatitis C virus (HCV) Core levels via ubiquitin-dependent proteasomal degradation. For this effect, ATRA downregulated both protein and enzyme activity levels of DNA methyltransferase 1 and 3b and activated E6AP expression via promoter hypomethylation in HepG2 cells but not in Hep3B cells, in which p53 was absent. Ectopic p53 expression but not E6AP overexpression restored the ability of ATRA to downregulate HCV Core levels in Hep3B cells, suggesting a direct role of p53 in the E6AP-mediated ubiquitination of HCV Core. ATRA also downregulated HCV Core levels during HCV infection in Huh7D cells to inhibit virus replication, providing theoretical basis for the clinical application of ATRA against HCV infection.

Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy.

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients.

Photoactive "Bionic Virus" Robustly Elicits the Synergy Anticancer Activity of Immunophotodynamic Therapy.

Coronavirus represents an inspiring model for designing drug delivery systems due to its unique infection machinery mechanism. Herein, we have developed a biomimetic viruslike nanocomplex, termed SDN, for improving cancer theranostics. SDN has a unique core-shell structure consisting of photosensitizer chlorin e6 (Ce6)-loaded nanostructured lipid carrier (CeNLC) (virus core)@poly(allylamine hydrochloride)-functionalized MnO2 nanoparticles (virus spike), generating a virus-mimicking nanocomplex. SDN not only prompted cellular uptake through rough-surface-mediated endocytosis but also achieved mitochondrial accumulation by the interaction of cationic spikes and the anionic mitochondrial surface, leading to mitochondria-specific photodynamic therapy. Meanwhile, SDN could even mediate oxygen generation to relieve tumor hypoxia and, consequently, improve macrophage-associated anticancer immune response. Importantly, SDN served as a robust magnetic resonance imaging (MRI) contrast agent due to the fast release of Mn2+ in the presence of intracellular redox components. We identified that SDN selectively accumulated in tumors and released Mn2+ to generate a 5.71-fold higher T1-MRI signal, allowing for effectively detecting suspected tumors. Particularly, SDN induced synergistic immunophotodynamic effects to eliminate malignant tumors with minimal adverse effects. Therefore, we present a novel biomimetic strategy for improving targeted theranostics, which has a wide range of potential biomedical applications.

Bioengineering of virus-like particles as dynamic nanocarriers for in vivo delivery and targeting to solid tumours.

Virus-like particles (VLPs) are known as self-assembled, non-replicative and non-infectious protein particles, which imitate the formation and structure of original wild type viruses, however, lack the viral genome and/or their fragments. The capacity of VLPs to encompass small molecules like nucleic acids and others has made them as novel vessels of nanocarriers for drug delivery applications. In addition, VLPs surface have the capacity to achieve variation of the surface display via several modification strategies including genetic modification, chemical modification, and non-covalent modification. Among the VLPs nanocarriers, Hepatitis B virus core (HBc) particles have been the most encouraging candidate. HBc particles are hollow nanoparticles in the range of 30-34nm in diameter and 7nm thick envelopes, consisting of 180 or 240 copies of identical polypeptide monomer. They also employ a distinctive position among the VLPs carriers due to the high-level synthesis, which serves as a strong protective capsid shell and efficient self-assembly properties. This review highlights on the bioengineering of HBc particles as dynamic nanocarriers for in vivo delivery and specific targeting to solid tumours.

Pharmacomodulation of a ligand targeting the HBV capsid hydrophobic pocket.

Hepatitis B virus (HBV) is a small enveloped retrotranscribing DNA virus and an important human pathogen. Its capsid-forming core protein (Cp) features a hydrophobic pocket proposed to be central notably in capsid envelopment. Indeed, mutations in and around this pocket can profoundly modulate, and even abolish, secretion of enveloped virions. We have recently shown that Triton X-100, a detergent used during Cp purification, binds to the hydrophobic pocket with micromolar affinity. We here performed pharmacomodulation of pocket binders through systematic modifications of the three distinct chemical moieties composing the Triton X-100 molecule. Using NMR and ITC, we found that the flat aromatic moiety is essential for binding, while the number of atoms of the aliphatic chain modulates binding affinity. The hydrophilic tail, in contrast, is highly tolerant to changes in both length and type. Our data provide essential information for designing a new class of HBV antivirals targeting capsid–envelope interactions.

A molecular simulation study of hepatitis B virus core protein and the nuclear protein allosteric modulators of phthalazinone derivatives.

Hepatitis B virus, causing hepatitis, cirrhosis, liver failure, and liver cancer, poses a serious threat to human health, and the currently approved drugs still cannot eliminate the virus completely. HBV core protein allosteric modulators (CpAMs) with a phthalazinone structure which targets the HBV core (HBc) protein have been seen as a new kind of drug because of their excellent antiviral effects. This study explores the structure-activity relationship and binding mechanism of phthalazinone molecules through three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, molecular dynamics, and binding free energy calculation and decomposition studies. In addition, CoMFA and CoMSIA models revealed that the steric field, the hydrophobic field, and the hydrogen bond acceptor field may play important roles in the binding process. The molecular docking and dynamics disclosed the most likely binding pose of phthalazinone derivatives with the HBc protein. The binding free energy calculation and decomposition analysis indicated that the van der Waals force was the driving force and that ValE124, ThrD109, ThrE128, LeuD140, IleD105, PheD110, ThrD33, and TrpD102 were the key residues. This study provides an important theoretical basis for the design and optimization of phthalazinone compounds.

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.