scholarly journals Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt HBV Capsids

2021 ◽  
Author(s):  
Christine Kim ◽  
Lauren F. Barnes ◽  
Christopher J. Schlicksup ◽  
Angela J. Patterson ◽  
Brian B. Bothner ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Core Protein ◽  
Innate Immune ◽  
Host Protein ◽  
Structural Proteins ◽  
Nuclear Trafficking ◽  
Terminal Domain ◽  
Viral Lifecycle ◽  
Capsid Stability

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Impβ) via Cp’s C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro . We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to “flip” to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.

scholarly journals Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt HBV Capsids

2021 ◽  
Author(s):  
Christine Kim ◽  
Lauren Barnes ◽  
Christopher Schlicksup ◽  
Angela Patterson ◽  
Brian Bothner ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Core Protein ◽  
Innate Immune ◽  
Host Protein ◽  
Structural Proteins ◽  
Nuclear Trafficking ◽  
Terminal Domain ◽  
Viral Lifecycle ◽  
Capsid Stability

ABSTRACTViral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Impβ) via Cp’s C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Impβ and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Impβ synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Impβ. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses.IMPORTANCEThe HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to “flip” to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.

scholarly journals Phosphorylation of the Arginine-Rich C-Terminal Domains of the Hepatitis B Virus (HBV) Core Protein as a Fine Regulator of the Interaction between HBc and Nucleic Acid

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 738 ◽  
Author(s):  
Hugues de Rocquigny ◽  
Virgile Rat ◽  
Florentin Pastor ◽  
Jean Luc Darlix ◽  
Christophe Hourioux ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleic Acids ◽  
Core Protein ◽  
Cell Trafficking ◽  
Terminal Domain ◽  
Capsid Shell ◽  
B Virus ◽  
Capsid Stability ◽  
Terminal Domains

The morphogenesis of Hepatitis B Virus (HBV) viral particles is nucleated by the oligomerization of HBc protein molecules, resulting in the formation of an icosahedral capsid shell containing the replication-competent nucleoprotein complex made of the viral polymerase and the pre-genomic RNA (pgRNA). HBc is a phospho-protein containing two distinct domains acting together throughout the viral replication cycle. The N-terminal domain, (residues 1–140), shown to self-assemble, is linked by a short flexible domain to the basic C-terminal domain (residues 150–183) that interacts with nucleic acids (NAs). In addition, the C-terminal domain contains a series of phospho-acceptor residues that undergo partial phosphorylation and de-phosphorylation during virus replication. This highly dynamic process governs the homeostatic charge that is essential for capsid stability, pgRNA packaging and to expose the C-terminal domain at the surface of the particles for cell trafficking. In this review, we discuss the roles of the N-terminal and C-terminal domains of HBc protein during HBV morphogenesis, focusing on how the C-terminal domain phosphorylation dynamics regulate its interaction with nucleic acids throughout the assembly and maturation of HBV particles.

scholarly journals Control of Alphavirus Replication in Neurons

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 78
Author(s):  
Jane Yeh ◽  
Elizabeth Troisi ◽  
Rachy Abraham ◽  
Diane Griffin
Keyword(s):  
Sindbis Virus ◽  
Regulatory Protein ◽  
Host Protein ◽  
Structural Proteins ◽  
Subgenomic Rna ◽  
Macro Domain ◽  
Mature Neurons ◽  
Host Protein Synthesis ◽  
Viral Structural Proteins

Sindbis virus causes age-dependent encephalomyelitis in mice. Young mice and immature neurons replicate the virus to high titers and die from infection while older mice and mature neurons restrict replication and survive infection. Studies to identify factors that affect maturation-dependent virus replication in neurons have used AP-7 rat olfactory neuronal cells that can be differentiated in vitro, and have identified roles for host transcription factors interferon regulatory protein (IRF) 7 and NF-B and viral proteins E2 and nsP3. IRF7 is required for neuronal survival and deficiency leads to paralysis and death of 4–6 weeks old C57BL/6 mice. Activation of NF-κB works in conjunction with PKR to facilitate replication in mature neurons by promoting shut-off of host protein synthesis and increasing translation of the viral structural proteins from subgenomic RNA. The macro domain of nsP3 binds and hydrolyses ADP-ribose from ADP-ribosylated proteins and is important for initiation of neuronal infection and for synthesis of viral structural proteins. Thus, neurons regulate translation of the structural proteins from subgenomic RNA required for the production of the infectious virus.

scholarly journals Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A)

2007 ◽  
Vol 403 (3) ◽  
pp. 553-563 ◽  
Author(s):  
Joji Iida ◽  
Krista L. Wilhelmson ◽  
Janet Ng ◽  
Peter Lee ◽  
Charlotte Morrison ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chondroitin Sulfate ◽  
Catalytic Domain ◽  
Core Protein ◽  
Active Form ◽  
Terminal Domain ◽  
Direct Binding ◽  
Membrane Type ◽  
Type 3

We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with βDX (p-nitro-β-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.

scholarly journals Preclinical Profile and Characterization of the Hepatitis B Virus Core Protein Inhibitor ABI-H0731

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Qi Huang ◽  
Dawei Cai ◽  
Ran Yan ◽  
Lichun Li ◽  
Yuhua Zong ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Dna Replication ◽  
Hepatitis B ◽  
Mechanism Of Action ◽  
De Novo ◽  
Core Protein ◽  
Protein Inhibitor ◽  
Circular Dna ◽  
B Virus

ABSTRACT ABI-H0731, a first-generation hepatitis B virus (HBV) core protein inhibitor, has demonstrated effective antiviral activity in chronic hepatitis B (CHB) patients in a phase 1b clinical trial and is currently being further evaluated in phase 2 clinical trials. Here, we report the preclinical profile of ABI-H0731. In in vitro cell culture systems (HepG2-derived cell lines HepAD38 and HepG2-NTCP and primary human hepatocytes [PHHs]), ABI-H0731 exhibited selective inhibition of HBV DNA replication (50% effective concentration [EC50] from 173 nM to 307 nM). Most importantly, ABI-H0731 suppressed covalently closed circular DNA (cccDNA) formation in two de novo infection models with EC50s from 1.84 μM to 7.3 μM. Mechanism-of-action studies indicated that ABI-H0731 is a direct-acting antiviral that targets HBV core protein, preventing HBV pregenomic RNA (pgRNA) encapsidation and subsequent DNA replication. The combination of ABI-H0731 with entecavir appears to decrease viral DNA faster and deeper than nucleoside/nucleotide analogue (NrtI) therapy alone. In addition, ABI-H0731 disrupts incoming nucleocapsids, causing the premature release of relaxed circular DNA (rcDNA) before delivery to the nucleus, and thus prevents new cccDNA formation. ABI-H0731 exhibits pangenotypic activity and is additive to moderately synergistic when combined with an NrtI. In addition to its potency and novel mechanism of action, ABI-H0731 possesses drug-like properties and a preclinical pharmacokinetic profile supportive of once-daily dosing in patients with CHB. Taken together, these data support the ongoing clinical development of ABI-H0731 as a treatment for HBV.

The Effect of Dipyridamole and RA 233 on Human Platelet Function in Vitro

1973 ◽  
Vol 29 (03) ◽  
pp. 694-700 ◽  
Author(s):  
Paul L. Rifkin ◽  
Marjorie B. Zucker
Keyword(s):  
Mechanism Of Action ◽  
Human Blood ◽  
Glass Bead ◽  
Heart Valves ◽  
Human Platelet ◽  
Drug Effects ◽  
Simple Relation ◽  
Prosthetic Heart Valves ◽  
Induced Aggregation

SummaryDipyridamole (Persantin) is reported to prolong platelet survival and inhibit embolism in patients with prosthetic heart valves, but its mechanism of action is unknown. Fifty jxM dipyridamole failed to reduce the high percentage of platelets retained when heparinized human blood was passed through a glass bead column, but prolonged the inhibition of retention caused by disturbing blood in vitro. Possibly the prostheses act like disturbance. Although RA 233 was as effective as dipyridamole in inhibiting the return of retention, it was less effective in preventing the uptake of adenosine into erythrocytes, and more active in inhibiting ADP-induced aggregation and release. Thus there is no simple relation between these drug effects.

scholarly journals In vitro activities of novel catecholate siderophores against Plasmodium falciparum.

1996 ◽  
Vol 40 (9) ◽  
pp. 2094-2098 ◽  
Author(s):  
B Pradines ◽  
F Ramiandrasoa ◽  
L K Basco ◽  
L Bricard ◽  
G Kunesch ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Mechanism Of Action ◽  
Ribonucleotide Reductase ◽  
Antimalarial Activity ◽  
Iron Chelators ◽  
Resistant Clone ◽  
Iron Deprivation ◽  
Level Of Activity ◽  
Susceptible Clone

The activities of novel iron chelators, alone and in combination with chloroquine, quinine, or artemether, were evaluated in vitro against susceptible and resistant clones of Plasmodium falciparum with a semimicroassay system. N4-nonyl,N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound 7) demonstrated the highest level of activity: 170 nM against a chloroquine-susceptible clone and 1 microM against a chloroquine-resistant clone (50% inhibitory concentrations). Compounds 6, 8, and 10 showed antimalarial activity with 50% inhibitory concentrations of about 1 microM. Compound 7 had no effect on the activities of chloroquine, quinine, and artemether against either clone, and compound 8 did not enhance the schizontocidal action of either chloroquine or quinine against the chloroquine-resistant clone. The incubation of compound 7 with FeCI3 suppressed or decreased the in vitro antimalarial activity of compound 7, while no effect was observed with incubation of compound 7 with CuSO4 and ZnSO4. These results suggest that iron deprivation may be the main mechanism of action of compound 7 against the malarial parasites. Chelator compounds 7 and 8 primarily affected trophozoite stages, probably by influencing the activity of ribonucleotide reductase, and thus inhibiting DNA synthesis.

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