ABSTRACT
Nucleoprotein (NP) is a key structural protein of influenza ribonucleoprotein complexes and is central to viral RNA packing and trafficking. NP also determines the sensitivity of influenza to myxovirus resistance protein 1 (MxA), an innate immunity factor that restricts influenza replication. A few critical MxA-resistant mutations have been identified in NP, including the highly conserved proline-283 substitution. This essential proline-283 substitution impairs influenza growth, a fitness defect that becomes particularly prominent at febrile temperature (39°C) when host chaperones are depleted. Here, we biophysically characterize proline-283 NP and serine-283 NP to test whether the fitness defect is caused by the proline-283 substitution introducing folding defects. We show that the proline-283 substitution changes the folding pathway of NP, making NP more aggregation prone during folding, but does not alter the native structure of the protein. These findings suggest that influenza has evolved to hijack host chaperones to promote the folding of otherwise biophysically incompetent viral proteins that enable innate immune system escape.
Subject(s)
Influenza, Human , Humans , Viral Core Proteins/genetics , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , RNA-Binding Proteins/metabolism , Nucleocapsid Proteins/metabolism , Myxovirus Resistance ProteinsABSTRACT
Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.
Subject(s)
Cell Membrane , Ebolavirus , Virus Assembly , Virus Release , Humans , Amino Acid Substitution , Cell Membrane/metabolism , Ebolavirus/metabolism , Ebolavirus/genetics , HEK293 Cells , Hemorrhagic Fever, Ebola/metabolism , Hemorrhagic Fever, Ebola/virology , Mutation , Nucleoproteins , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylserines/metabolism , Phosphatidylserines/chemistry , Protein Binding , Static Electricity , Viral Core Proteins/metabolism , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Matrix Proteins/genetics , Viral Matrix Proteins/chemistry , Virion/metabolism , Virion/geneticsABSTRACT
Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 via convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.
Subject(s)
Hepatitis B , Nucleic Acids , Humans , Capsid/chemistry , Capsid Proteins/metabolism , Hepatitis B virus , Hepatitis B/metabolism , Viral Core Proteins/analysis , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Virus Replication , Antiviral Agents/metabolismABSTRACT
Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.
Subject(s)
Cryoelectron Microscopy , Models, Molecular , Protein Multimerization , Vaccinia virus/ultrastructure , Vaccinia virus/chemistry , Vaccinia virus/metabolism , Poxviridae/ultrastructure , Poxviridae/metabolism , Poxviridae/chemistry , Viral Core Proteins/chemistry , Viral Core Proteins/ultrastructure , Viral Core Proteins/metabolism , HumansABSTRACT
Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.
Subject(s)
G-Quadruplexes , Hepatitis B virus , Hepatitis B , Humans , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Hepatitis B/virology , Hepatitis B e Antigens/metabolism , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Virus Replication/genetics , Cell Line , G-Quadruplexes/drug effects , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , Mutation , Aminoquinolines/pharmacologyABSTRACT
The Hepatitis B virus (HBV) core protein is an attractive target for preventing capsid assembly and viral replication. Drug repurposing strategies have introduced several drugs targeting HBV core protein. This study used a fragment-based drug discovery (FBDD) approach to reconstruct a repurposed core protein inhibitor to some novel antiviral derivatives. Auto Core Fragment in silico Screening (ACFIS) server was used for deconstruction-reconstruction of Ciclopirox in complex with HBV core protein. The Ciclopirox derivatives were ranked based on their free energy of binding (ΔGB). A quantitative structure affinity relationship (QSAR) was established on the Ciclopirox derivatives. The model was validated by a Ciclopirox-property-matched decoy set. A principal component analysis (PCA) was also assessed to define the relationship of the predictive variable of the QSAR model. 24-derivatives with a ΔGB (-16.56±1.46 Kcal.mol-1) more than Ciclopirox was highlighted. A QSAR model with a predictive power of 88.99% (F-statistics = 9025.78, corrected df(25), Pr > F = 0.0001) was developed by four predictive descriptors (ATS1p, nCs, Hy, F08[C-C]). The model validation showed no predictive power for the decoy set (Q2 = 0). No significant correlation was observed between predictors. By directly attaching to the core protein carboxyl-terminal domain, Ciclopirox derivatives may be able to suppress HBV virus assembly and subsequent viral replication inhibition. Hydrophobic residue Phe23 is a critical amino acid in the ligand binding domain. These ligands share the same physicochemical properties that lead to the development of a robust QSAR mode. The same strategy may also be used for future drug discovery of viral inhibitors.
Subject(s)
Hepatitis B , Virus Assembly , Humans , Hepatitis B virus/metabolism , Ciclopirox/pharmacology , Virus Replication , Antiviral Agents/chemistry , Capsid Proteins/metabolism , Drug Discovery , Viral Core Proteins/chemistryABSTRACT
Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane. IPs participate in a number of cellular pathways including immune response and surveillance, and cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates, and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in the production of amyloid-ß plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.
Subject(s)
Aspartic Acid Proteases , Bacterial Proteins , Lipids , Methanomicrobiaceae , Presenilins , Aspartic Acid Proteases/chemistry , Lipids/chemistry , Presenilins/chemistry , Methanomicrobiaceae/chemistry , Bacterial Proteins/chemistry , Viral Core Proteins/chemistry , KineticsABSTRACT
Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.
Subject(s)
Amino Acid Substitution , Conserved Sequence , DNA, Circular , Hepatitis B Core Antigens , Hepatitis B virus , Hepatitis B , Lysine , Viral Core Proteins , Amino Acid Sequence , Conserved Sequence/genetics , DNA, Circular/biosynthesis , DNA, Circular/genetics , DNA, Circular/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Hepatitis B/virology , Hepatitis B Core Antigens/chemistry , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/metabolism , Hepatitis B virus/chemistry , Hepatitis B virus/genetics , Hepatitis B virus/growth & development , Hepatitis B virus/metabolism , Humans , Lysine/genetics , Lysine/metabolism , Mutation , Nucleocapsid/metabolism , Polyadenylation/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Virion/growth & development , Virus Replication/geneticsABSTRACT
Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Damage , Hepatitis B virus/physiology , Hepatocytes/virology , Viral Core Proteins/metabolism , Amino Acid Motifs , Checkpoint Kinase 2/metabolism , Cytoplasm/metabolism , Cytoplasm/virology , Etoposide/pharmacology , Hep G2 Cells , Hepatitis B e Antigens/metabolism , Hepatitis B virus/drug effects , Humans , Hydrogen Peroxide/pharmacology , Phosphorylation , Serine/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Viral Core Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Virus Replication/drug effectsABSTRACT
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.
Subject(s)
Hepatitis B e Antigens/metabolism , Hepatitis B virus/physiology , Hepatitis B/virology , Protein Multimerization , Viral Core Proteins/chemistry , Virus Assembly , Virus Replication , DNA Replication , DNA, Viral , Hep G2 Cells , Humans , Nucleocapsid , Viral Core Proteins/metabolismABSTRACT
Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a â¼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
Subject(s)
Bacteriophage T7/genetics , DNA, Viral/chemistry , Periplasm/chemistry , Viral Core Proteins/chemistry , Computational Biology , Cryoelectron Microscopy , Cytoplasm/chemistry , DNA, Viral/metabolism , Lipid Bilayers/metabolism , Periplasm/genetics , Periplasm/metabolism , Podoviridae/chemistry , Podoviridae/genetics , Viral Core Proteins/metabolismABSTRACT
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.
Subject(s)
Antiviral Agents/pharmacology , Capsid/metabolism , Hepatitis B virus/drug effects , Hepatitis B virus/physiology , Nucleocapsid/metabolism , RNA/metabolism , Viral Core Proteins/genetics , Virus Assembly/physiology , DNA, Viral , Hep G2 Cells , Hepatitis B virus/chemistry , Hepatitis B virus/genetics , Humans , RNA/genetics , RNA, Viral/genetics , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Virus Assembly/geneticsABSTRACT
The Ebola virus matrix protein VP40 forms distinct structures linked to distinct functions in the virus life cycle. Dimeric VP40 is a structural protein associated with virus assembly, while octameric, ring-shaped VP40 is associated with transcriptional control. In this study, we show that suitable nucleic acid is sufficient to trigger a dynamic transformation of VP40 dimer into the octameric ring. Deep sequencing reveals a binding preference of the VP40 ring for the 3' untranslated region of cellular mRNA and a guanine- and adenine-rich binding motif. Complementary analyses of the nucleic-acid-induced VP40 ring by native mass spectrometry, electron microscopy, and X-ray crystal structures at 1.8 and 1.4 Å resolution reveal the stoichiometry of RNA binding, as well as an interface involving a key guanine nucleotide. The host factor-induced structural transformation of protein structure in response to specific RNA triggers in the Ebola virus life cycle presents unique opportunities for therapeutic inhibition.
Subject(s)
3' Untranslated Regions , Ebolavirus/genetics , Guanine/chemistry , Host-Pathogen Interactions/genetics , Nucleoproteins/chemistry , Viral Core Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Ebolavirus/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Guanine/metabolism , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Models, Molecular , Nucleoproteins/genetics , Nucleoproteins/metabolism , Nucleotide Motifs , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Virus Assembly/genetics , Virus Release/geneticsABSTRACT
Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and, furthermore, is incorporated into the viral capsid, accounting for most of the "endogenous kinase" activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs in the HBc NTD known as cyclin docking motifs (CDMs), which mediate the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection.IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.
Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Hepatitis B virus/physiology , Protein Interaction Domains and Motifs , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , Virus Replication , Capsid/enzymology , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cyclins/metabolism , Hep G2 Cells , Hepatitis B virus/chemistry , Humans , Nucleocapsid/metabolism , Phosphorylation , Protein Domains , Protein Interaction Domains and Motifs/genetics , RNA, Viral/metabolism , Viral Core Proteins/genetics , Virus AssemblyABSTRACT
Photodynamic therapy and adipose browning induction are two promising approaches to reverse obesity. The former strategy acts rapidly and locally, whereas the latter has a more gradual and widespread effect. Despite their complementarity, they have rarely been combined and imaged non-invasively in vivo. Here we introduce an adipose-targeting hepatitis B core protein complex that contains a traceable photosensitizer (ZnPcS4 (zinc phthalocyanine tetrasulfonate)) and a browning agent (rosiglitazone) that allows simultaneous photodynamic and browning treatments, with photoacoustic molecular imaging. After intravenous injection in obese mice, the complex binds specifically to white adipose tissues, especially those rich in blood supply, and drives adipose reduction thanks to the synergy of ZnPcS4 photodynamics and rosiglitazone browning. Using photoacoustic molecular imaging, we could monitor the changes induced by the treatment, which included complex activity, lipid catabolism and angiogenesis. Our findings demonstrate the anti-obesity potential of our feedback-based synergic regimen orchestrated by the targeted hepatitis B core complex.
Subject(s)
Adipose Tissue, White/drug effects , Obesity/therapy , Photoacoustic Techniques , Viral Core Proteins/chemistry , Adipose Tissue, White/diagnostic imaging , Adipose Tissue, White/metabolism , Animals , Hepatitis B/genetics , Humans , Indoles/chemistry , Indoles/pharmacology , Mice , Molecular Imaging/methods , Obesity/metabolism , Obesity/pathology , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Rosiglitazone/pharmacology , Viral Core Proteins/pharmacologyABSTRACT
Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication.
Subject(s)
Capsid Proteins/chemistry , Hepatitis B virus/genetics , Hepatitis B/virology , Viral Core Proteins/chemistry , Virus Replication , Amino Acid Motifs , Animals , Capsid Proteins/genetics , Hep G2 Cells , Hepatitis B Core Antigens/chemistry , Hepatitis B Core Antigens/genetics , Hepatitis B virus/physiology , Humans , Phosphorylation , Plasmids , Protein Binding , Protein Domains , Protein Phosphatase 2/metabolism , RNA, Viral/genetics , Rabbits , Viral Core Proteins/genetics , VirionABSTRACT
East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Nanotechnology/methods , Protozoan Vaccines/immunology , Theileria parva/physiology , Theileriasis/immunology , Tick-Borne Diseases/immunology , Animals , Antibodies, Protozoan/blood , Cattle , Hepatitis B virus/chemistry , Hepatitis B virus/genetics , Mice , Mineral Oil/administration & dosage , Nanoparticles/chemistry , Protozoan Proteins/genetics , Protozoan Vaccines/genetics , RAW 264.7 Cells , Silicon Dioxide/chemistry , Ticks , Vaccination , Vaccines, Subunit , Viral Core Proteins/chemistry , Viral Core Proteins/geneticsABSTRACT
Ebola filovirus (EBOV) is one of the deadliest known infectious agents, and a cause of Western African epidemics from 2013 to 2016. The virus has infected nearly 3000 humans and almost 1900 have died. In the past few years, various small molecules have been discovered to display efficiency against EBOV and some of them have progressed towards clinical trials. Even though continuous attempts have been made to find antiEBOV therapeutics, no potential drugs are yet approved against this viral infection. The development of small antiviral inhibitors has gained tremendous attention in the attempt to overcome EVD. With this background, we seek to offer molecular insights into EBOV VP40 protein inhibition, using all atom molecular mechanics methodology and binding free energy calculations. We have selected five novel reported inhibitors against VP40 protein, namely Comp1, Comp2, Comp3, Comp4, and Comp5, and explored their binding against the same target. It was evident from the analysis that all the inhibitors displayed stability in complex with VP40 protein; however, Comp1 exhibited enhanced stability and compactness. Comp1 unveiled favorable binding, which accounted for positive correlation motions in the active site residues. Likewise, Comp1 revealed the most promising binding (ΔGbind - 40.3504 kcal/mol) as compared to the other four inhibitors, which disclosed relatively less favorable ΔGbind. The highest binding energy of Comp1 to VP40 protein can be primarily endorsed to the upsurge in van der Waals energy by ΔEvdW - 37.1609 kcal/mol and Coulomb energy by ΔEele - 52.7332 kcal/mol. Also, the hydrogen bond network is robust in Comp1-VP40 complex, with four hydrogen bonds, whilst it is less in other inhibitors. The outcomes from this report may assist in the advancement of novel VP40 inhibitors with high selectivity and potency for EVD therapeutics.
Subject(s)
Molecular Dynamics Simulation , Nucleoproteins/antagonists & inhibitors , Nucleoproteins/chemistry , Viral Core Proteins/antagonists & inhibitors , Viral Core Proteins/chemistry , Amino Acids/metabolism , Humans , Inhibitory Concentration 50 , Ligands , Molecular Docking Simulation , Protein Stability , RNA/chemistry , RNA/metabolism , ThermodynamicsABSTRACT
Non-enveloped Nackednaviridae and enveloped hepadnaviridae both have capsids that are formed by related small proteins which evolved more than 430 Mya. In Hepatitis B virus, which belongs to the enveloped hepadnaviridae, this small protein is termed Hepatitis B core protein (Hbc). Its function, as building block of a major human pathogen, triggered extensive research that elucidated the importance of almost every single amino acid for the structural integrity of the capsids and the orderly progression of the viral life cycle. In particular, encapsidation of the genome, envelopment of the capsid, uncoating of the genome and targeting of the different compartments during viral maturation have been a vivid focus of research. HBc has also been developed as a biotechnological tool for the design of nano-containers with tailored properties. These nano-containers can display foreign epitopes on their surfaces and induce a strong immune response, which is attractive for the development of vaccines against other pathogens. This chapter will discuss some of the unique properties of HBc and their significance for the formation of a functional macromolecular capsid.
Subject(s)
Capsid/chemistry , Hepatitis B virus/chemistry , Viral Core Proteins/chemistry , Capsid Proteins/chemistry , Hepatitis B/virology , HumansABSTRACT
The viral ribonucleoprotein (vRNP) of the influenza A virus (IAV) is responsible for the viral RNA transcription and replication in the nucleus, and its functions rely on host factors. Previous studies have indicated that eukaryotic translation elongation factor 1 delta (eEF1D) may associate with RNP subunits, but its roles in IAV replication are unclear. Herein, we showed that eEF1D was an inhibitor of IAV replication because knockout of eEF1D resulted in a significant increase in virus yield. eEF1D interacted with RNP subunits polymerase acidic protein (PA), polymerase basic 1 (PB1), polymerase basic 2 (PB2), and also with nucleoprotein (NP) in an RNA-dependent manner. Further studies revealed that eEF1D impeded the nuclear import of NP and PA-PB1 heterodimer of IAV, thereby suppressing the vRNP assembly, viral polymerase activity, and viral RNA synthesis. Together, our studies demonstrate eEF1D negatively regulating the IAV replication by inhibition of the nuclear import of RNP subunits, which not only uncovers a novel role of eEF1D in IAV replication but also provides new insights into the mechanisms of nuclear import of vRNP proteins.IMPORTANCE Influenza A virus is the major cause of influenza, a respiratory disease in humans and animals. Different from most other RNA viruses, the transcription and replication of IAV occur in the cell nucleus. Therefore, the vRNPs must be imported into the nucleus for viral transcription and replication, which requires participation of host proteins. However, the mechanisms of the IAV-host interactions involved in nuclear import remain poorly understood. Here, we identified eEF1D as a novel inhibitor for the influenza virus life cycle. Importantly, eEF1D impaired the interaction between NP and importin α5 and the interaction between PB1 and RanBP5, which impeded the nuclear import of vRNP. Our studies not only reveal the molecular mechanisms of the nuclear import of IAV vRNP but also provide potential anti-influenza targets for antiviral development.