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Capsid assembly modulators (CAMs) have the potential to cure chronic hepatitis B, as demonstrated in clinical trials. Lead compounds NVR3-778 and 5a were found to exist in normal and flipped conformations through induced fit docking. Therefore, we designed and synthesized series I and II compounds by interchanging the amide and sulfonamide bonds of 5a to modify both the tolerance region and solvent-opening region. Among them, compound 4a (EC50 = 0.24 ± 0.10 µM, CC50 > 100 µM) exhibited potent anti-HBV activity with low toxicity, surpassing the lead compounds NVR3-778 (EC50 = 0.29 ± 0.03 µM, CC50 = 20.78 ± 2.29 µM) and 5a (EC50 = 0.50 ± 0.07 µM, CC50 = 48.16 ± 9.15 µM) in HepAD38 cells. Additionally, compared with the lead compound, 4a displayed a stronger inhibitory effect on HBV capsid protein assembly. Molecular dynamics (MD) simulations confirmed that the normal conformation of 4a had relatively stable conformation at different frames of binding modes. Furthermore, 4a showed better metabolic stability in human plasma than positive control drugs. Therefore, compound 4a could be further structurally modified as a potent lead compound.
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Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.
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Cápside , Proteínas Fluorescentes Verdes , Virus de la Hepatitis B , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Cápside/química , Cápside/metabolismo , Ensamble de Virus , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Virión/metabolismo , Virión/química , Propiedades de SuperficieRESUMEN
The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.
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Secuencias de Aminoácidos , Virus BK , Proteínas de la Cápside , Vesículas Extracelulares , Virión , Ensamble de Virus , Virus BK/genética , Virus BK/fisiología , Virus BK/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virología , Humanos , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Virión/metabolismo , Virión/genética , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HEK293RESUMEN
We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.
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Antivirales , Antivirales/farmacología , Antivirales/química , Humanos , Animales , Proteínas 14-3-3/metabolismo , Complejos Multiproteicos/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Línea CelularRESUMEN
The hepatitis B virus (HBV) capsid or core protein is a promising drug target currently being investigated for potential curative therapies for chronic HBV infection. In this study, we performed extensive in vitro and in vivo characterization of a novel and potent HBV core protein assembly modulator (CpAM), CU15, for both anti-HBV activity and druggability properties. CU15 potently inhibited HBV DNA replication in in vitro HBV-infected HepG2.2.15 cells (EC50 of 8.6 nM), with a low serum shift. It was also effective in inhibiting HBV DNA and cccDNA formation in de novo HBV-infected primary human hepatocytes. Furthermore, CU15 was active across several HBV genotypes and across clinically relevant core protein variants. After oral administration to an in vivo HBV mouse model, CU15 significantly reduced plasma HBV DNA and RNA levels, at plasma exposure consistent with the estimated in vitro potency. In vitro, CU15 exhibited excellent passive permeability and relatively high metabolic stability in liver preparations across species (human > dog> rat). In vitro human liver microsomal studies suggest that the compound's major metabolic pathway is CYP3A-mediated oxidation. Consistent with the in vitro findings, CU15 is a compound with a low-to-moderate clearance and high oral bioavailability in rats and dogs. Based on the apparent in vitro-in vivo correlation observed, CU15 has the potential to exhibit low clearance and high oral bioavailability in humans. In addition, CU15 also showed low drug-drug interaction liability with an acceptable in vitro safety profile (IC50 > 10 µM).
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Antivirales , Virus de la Hepatitis B , Hepatitis B Crónica , Humanos , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/farmacocinética , Hepatitis B Crónica/tratamiento farmacológico , Virus de la Hepatitis B/efectos de los fármacos , Células Hep G2 , Perros , Masculino , Ratas , ADN Viral , Ratones , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Replicación Viral/efectos de los fármacos , Proteínas del Núcleo Viral/metabolismo , Ratas Sprague-DawleyRESUMEN
Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.
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Virus de la Hepatitis B , Hepatitis B Crónica , Hepatocitos , Humanos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Animales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Proteínas del Núcleo Viral/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Antígenos del Núcleo de la Hepatitis B/metabolismo , Cápside/metabolismo , Cápside/efectos de los fármacos , Hígado/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Ensamble de Virus/efectos de los fármacos , Apoptosis/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Adeno-associated Virus (AAV) is a promising vector for gene therapy. However, few studies have focused on producing virus-like particles (VLPs) of AAV in cells, especially in E. coli. In this study, we describe a method to produce empty VP3-only VLPs of AAV2 in E. coli by co-expressing VP3 and assembly-activating protein (AAP) of AAV2. Although the yields of VLPs produced with our method were low, the VLPs were able to self-assemble in E. coli without the need of in vitro capsid assembly. The produced VLPs were characterized by immunological detection and transmission electron microscopy (TEM). In conclusion, this study demonstrated that capsid assembly of AAV2 is possible in E. coli, and E. coli may be a candidate system for production of VLPs of AAV.
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Proteínas de la Cápside , Dependovirus , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Dependovirus/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/biosíntesis , Virión/genética , Virión/metabolismo , Ensamble de Virus , Vectores Genéticos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/química , Parvovirinae/genética , HumanosRESUMEN
BACKGROUND & AIMS: Functional cure for chronic hepatitis B (CHB) requires finite treatment. Two agents under investigation with the goal of achieving functional cure are the small-interfering RNA JNJ-73763989 (JNJ-3989) and the capsid assembly modulator JNJ-56136379 (JNJ-6379; bersacapavir). METHODS: REEF-2, a phase IIb, double-blind, placebo-controlled, randomized study, enrolled 130 nucleos(t)ide analogue (NA)-suppressed hepatitis B e-antigen (HBeAg)-negative patients with CHB who received JNJ-3989 (200 mg subcutaneously every 4 weeks) + JNJ-6379 (250 mg oral daily) + NA (oral daily; active arm) or placebos for JNJ-3989 and JNJ-6379 +active NA (control arm) for 48 weeks followed by 48 weeks off-treatment follow-up. RESULTS: At follow-up Week 24, no patients achieved the primary endpoint of functional cure (off-treatment hepatitis B surface antigen [HBsAg] seroclearance). No patients achieved functional cure at follow-up Week 48. There was a pronounced on-treatment reduction in mean HBsAg from baseline at Week 48 in the active arm vs. no decline in the control arm (1.89 vs. 0.06 log10 IU/ml; p = 0.001). At follow-up Week 48, reductions from baseline were >1 log10 IU/ml in 81.5% vs. 12.5% of patients in the active and control arms, respectively, and 38/81 (46.9%) patients in the active arm achieved HBsAg <100 IU/ml vs. 6/40 (15.0%) patients in the control arm. Off-treatment HBV DNA relapse and alanine aminotransferase increases were less frequent in the active arm with 7/77 (9.1%) and 11/41 (26.8%) patients in the active and control arms, respectively, restarting NAs during follow-up. CONCLUSIONS: Finite 48-week treatment with JNJ-3989 + JNJ-6379 + NA resulted in fewer and less severe post-treatment HBV DNA increases and alanine aminotransferase flares, and a higher proportion of patients with off-treatment HBV DNA suppression, with or without HBsAg suppression, but did not result in functional cure. IMPACT AND IMPLICATIONS: Achieving a functional cure from chronic hepatitis B (CHB) with finite treatments is a major unmet medical need. The current study assessed the rate of functional cure and clinical outcome after controlled nucleos(t)ide analogue (NA) withdrawal in patients with low levels of HBsAg induced by 48 weeks of treatment with the small-interfering RNA JNJ-3989 and the capsid assembly modulator JNJ-6379 plus NA vs. patients who only received NA treatment. Though functional cure was not achieved by any patient in either arm, the 48-week treatment regimen of JNJ-3989, JNJ-6379, and NA did result in more patients achieving pronounced reductions in HBsAg, with clinically meaningful reductions maintained for up to 48 weeks off all treatments, as well as fewer off-treatment HBV DNA increases and alanine aminotransferase flares. These findings provide valuable insights for future studies investigating potential finite treatment options, while the reported efficacy and safety outcomes may be of interest to healthcare providers making treatment decisions for patients with NA-suppressed HBeAg-negative CHB. GOV IDENTIFIER: NCT04129554.
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Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.
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Virus de la Lengua Azul , Proteínas de la Cápside , Cápside , Microscopía por Crioelectrón , ARN Viral , Empaquetamiento del Genoma Viral , Virus de la Lengua Azul/genética , Virus de la Lengua Azul/fisiología , Virus de la Lengua Azul/metabolismo , Cápside/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Animales , ARN Viral/metabolismo , ARN Viral/genética , Genoma Viral/genética , Ensamble de Virus , Tomografía con Microscopio Electrónico , Virión/metabolismo , Virión/genética , Virión/ultraestructura , Modelos Moleculares , Línea Celular , CricetinaeRESUMEN
Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a promising therapeutic approach for the treatment of HBV infection. In this study, the hit compound CDI (IC50 = 2.46 ± 0.33 µM) was identified by screening of an in-house compound library. And then novel potent benzimidazole derivatives were designed and synthesized as core assembly modulators, and their antiviral effects were evaluated in vitro and in vivo biological experiments. The results indicated that compound 26f displayed the most optimized modulator of HBV capsid assembly (IC50 = 0.51 ± 0.20 µM, EC50 = 2.24 ± 0.43 µM, CC50 = 84.29 µM) and high selectivity index. Moreover, treatment with compound 26f for 14 days significantly decreased serum levels of HBV DNA levels in the Hydrodynamic-Injection (HDI) mouse model. Therefore, compound 26f could be considered as a promising candidate drug for further development of novel HBV CAMs with the desired potency and safety.
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Antivirales , Bencimidazoles , Virus de la Hepatitis B , Hepatitis B , Bencimidazoles/química , Bencimidazoles/farmacología , Bencimidazoles/síntesis química , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Virus de la Hepatitis B/efectos de los fármacos , Animales , Ratones , Humanos , Hepatitis B/tratamiento farmacológico , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Cápside/efectos de los fármacos , Cápside/metabolismo , Pruebas de Sensibilidad Microbiana , Células Hep G2 , Desarrollo de MedicamentosRESUMEN
Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.
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Antivirales , Apoptosis , Regulación Viral de la Expresión Génica , Antígenos del Núcleo de la Hepatitis B , Virus de la Hepatitis B , Hepatocitos , Biosíntesis de Proteínas , Animales , Ratones , Antivirales/farmacología , Antivirales/uso terapéutico , Apoptosis/efectos de los fármacos , Cápside/química , Cápside/clasificación , Cápside/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Hepatitis B/tratamiento farmacológico , Hepatitis B/inmunología , Hepatitis B/metabolismo , Hepatitis B/virología , Antígenos del Núcleo de la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis B/patogenicidad , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Ratones Endogámicos C57BL , Ratones SCID , Replicación Viral , Línea Celular , Linfocitos T CD8-positivos/inmunología , Presentación de AntígenoRESUMEN
Recombinant adeno-associated virus (rAAV) is one of the prominent gene delivery vehicles that has opened promising opportunities for novel gene therapeutic approaches. However, the current major viral vector production platform, triple transfection in mammalian cells, may not meet the increasing demand. Thus, it is highly required to understand production bottlenecks from the host cell perspective and engineer the cells to be more favorable and tolerant to viral vector production, thereby effectively enhancing rAAV manufacturing. In this review, we provided a comprehensive exploration of the intricate cellular process involved in rAAV production, encompassing various stages such as plasmid entry to the cytoplasm, plasmid trafficking and nuclear delivery, rAAV structural/non-structural protein expression, viral capsid assembly, genome replication, genome packaging, and rAAV release/secretion. The knowledge in the fundamental biology of host cells supporting viral replication as manufacturing factories or exhibiting defending behaviors against viral production is summarized for each stage. The control strategies from the perspectives of host cell and materials (e.g., AAV plasmids) are proposed as our insights based on the characterization of molecular features and our existing knowledge of the AAV viral life cycle, rAAV and other viral vector production in the Human embryonic kidney (HEK) cells.
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Dependovirus , Mamíferos , Humanos , Animales , Dependovirus/genética , Citoplasma , TransfecciónRESUMEN
BACKGROUND/AIMS: Four-week treatment of linvencorvir (RO7049389) was generally safe and well tolerated, and showed anti-viral activity in chronic hepatitis B (CHB) patients. This study evaluated the efficacy, safety, and pharmacokinetics of 48-week treatment with linvencorvir plus standard of care (SoC) in CHB patients. METHODS: This was a multicentre, non-randomized, non-controlled, open-label phase 2 study enrolling three cohorts: nucleos(t)ide analogue (NUC)-suppressed patients received linvencorvir plus NUC (Cohort A, n=32); treatment-naïve patients received linvencorvir plus NUC without (Cohort B, n=10) or with (Cohort C, n=30) pegylated interferon-α (Peg-IFN-α). Treatment duration was 48 weeks, followed by NUC alone for 24 weeks. RESULTS: 68 patients completed the study. No patient achieved functional cure (sustained HBsAg loss and unquantifiable HBV DNA). By Week 48, 89% of treatment-naïve patients (10/10 Cohort B; 24/28 Cohort C) reached unquantifiable HBV DNA. Unquantifiable HBV RNA was achieved in 92% of patients with quantifiable baseline HBV RNA (14/15 Cohort A, 8/8 Cohort B, 22/25 Cohort C) at Week 48 along with partially sustained HBV RNA responses in treatment-naïve patients during follow-up period. Pronounced reductions in HBeAg and HBcrAg were observed in treatment-naïve patients, while HBsAg decline was only observed in Cohort C. Most adverse events were grade 1-2, and no linvencorvir-related serious adverse events were reported. CONCLUSION: 48-week linvencorvir plus SoC was generally safe and well tolerated, and resulted in potent HBV DNA and RNA suppression. However, 48-week linvencorvir plus NUC with or without Peg-IFN did not result in the achievement of functional cure in any patient.
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Antivirales , Hepatitis B Crónica , Imidazoles , Pirazinas , Humanos , Antivirales/efectos adversos , Cápside , ADN Viral , Antígenos e de la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Polietilenglicoles , ARN , Nivel de Atención , Resultado del TratamientoRESUMEN
Development of new anti-hepatitis B virus (HBV) drugs that target viral capsid assembly is a very active research field. We identify a novel phthalazinone derivative, compound 5832, as a potent HBV inhibitor. In this study, we intend to elaborate the antiviral effect and mechanism of 5832 against HBV in vitro and in vivo. Compound 5832 treatment induces the formation of genome-free empty capsid by interfering with the core protein assembly domain, which significantly decreases the extracellular and intracellular HBV DNA. In the AAV-HBV transduced mouse model, 5832 suppresses serum HBV DNA after 4-week treatment, and decreases HBsAg and HBeAg levels. 5832 treatment also reduces intrahepatic HBV RNA, DNA and HBcAg levels. During the follow-up period after treatment withdrawal, serum antigen levels demonstrated no increase. We demonstrate 5832 treatment could active apoptotic signaling by elevating the expression of death receptor 5 (DR5), which participated in corresponding HBcAg-positive hepatocyte eradication. Phthalazinone derivative 5832 may serve as a promising anti-HBV drug candidate to improve the treatment options for chronic HBV infection.
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Hepatitis B Crónica , Hepatitis B , Ratones , Animales , Virus de la Hepatitis B , Antígenos del Núcleo de la Hepatitis B/metabolismo , Cápside , ADN Viral/genética , Proteínas de la Cápside/metabolismo , Antígenos de Superficie de la Hepatitis B , Antivirales/uso terapéuticoRESUMEN
Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.
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Islas Genómicas , Fagos de Staphylococcus , Staphylococcus aureus , Humanos , Proteínas de la Cápside/química , Microscopía por Crioelectrón , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/virología , Fagos de Staphylococcus/genética , Factores de Virulencia/genética , Transducción Genética , Empaquetamiento del ADN , Conformación de Ácido NucleicoRESUMEN
Several evolved properties of adeno-associated virus (AAV), such as broad tropism and immunogenicity in humans, are barriers to AAV-based gene therapy. Most efforts to re-engineer these properties have focused on variable regions near AAV's 3-fold protrusions and capsid protein termini. To comprehensively survey AAV capsids for engineerable hotspots, we determined multiple AAV fitness phenotypes upon insertion of six structured protein domains into the entire AAV-DJ capsid protein VP1. This is the largest and most comprehensive AAV domain insertion dataset to date. Our data revealed a surprising robustness of AAV capsids to accommodate large domain insertions. Insertion permissibility depended strongly on insertion position, domain type, and measured fitness phenotype, which clustered into contiguous structural units that we could link to distinct roles in AAV assembly, stability, and infectivity. We also identified engineerable hotspots of AAV that facilitate the covalent attachment of binding scaffolds, which may represent an alternative approach to re-direct AAV tropism.
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Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal in solution and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.
RESUMEN
Small molecules that bind to the pocket targeted by a peptide, termed capsid assembly inhibitor (CAI), have shown antiviral effects with unique mechanisms of action. We report the discovery of two natural compounds, sennoside A (SA) and sennoside B (SB), derived from medicinal plants that bind to this pocket in the C-terminal domain of capsid (CA CTD). Both SA and SB were identified via a drug-screening campaign that utilized a time-resolved fluorescence resonance energy transfer assay. They inhibited the HIV-1 CA CTD/CAI interaction at sub-micromolar concentrations of 0.18 µM and 0.08 µM, respectively. Mutation of key residues (including Tyr 169, Leu 211, Asn 183, and Glu 187) in the CA CTD decreased their binding affinity to the CA monomer, from 1.35-fold to 4.17-fold. Furthermore, both compounds induced CA assembly in vitro and bound directly to the CA hexamer, suggesting that they interact with CA beyond the CA CTD. Molecular docking showed that both compounds were bound to the N-terminal domain (NTD)/CTD interface between adjacent protomers within the CA hexamer. SA established a hydrogen-bonding network with residues N57, V59, Q63, K70, and N74 of CA1-NTD and Q179 of CA2-CTD. SB formed hydrogen bonds with the N53, N70, and N74 residues of CA1-NTD, and the A177and Q179 residues of CA2-CTD. Both compounds, acting as glue, can bring αH4 in the NTD and αH9 in the CTD of the NTD/CTD interface close to each other. Collectively, our research indicates that SA and SB, which enhance CA assembly, could serve as novel chemical tools to identify agents that modulate HIV-1 CA assembly. These natural compounds may potentially lead to the development of new antiviral therapies with unique mechanisms of action.
RESUMEN
The core protein allosteric modulators (CpAMs) have shown great potential as highly effective antiviral drugs against hepatitis B virus (HBV) in preclinical studies and clinical trials. In this study, we evaluated a small molecule compound called QL-007, which could potentially influence capsid assembly, using HBV replicated and susceptible cell models as well as mice infected with rAAV-HBV. QL-007 significantly inhibited HBV replication in a dose-dependent manner both in vitro and in vivo, resulting in significant decreases in HBV DNA, 3.5 kb HBV RNA and HBeAg. Furthermore, QL-007 not only induced the formation of misshaped Cp149 capsids but also possessed the capability to disassemble HBV capsids. It is noteworthy that QL-007 effectively reduced cccDNA biosynthesis in de novo infections. Mechanistically, QL-007 blocked the encapsidation of pgRNA and induced aberrant polymers assembly at concentrations ≥100 nM, while having no impact on the stability of core proteins. In conclusion, our findings underscore the potential of QL-007 as an effective agent against HBV replication and introduce it as a novel CpAM for the antiviral treatment of chronic hepatitis B.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Animales , Ratones , Cápside , Ensamble de Virus , Proteínas del Núcleo Viral/genética , Proteínas de la Cápside/metabolismo , Antivirales/uso terapéutico , Replicación ViralRESUMEN
IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.