Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids

AbstractThe icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid-like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C-terminal basic domain by the 17 kDa Staphylococcus aureus nuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild-type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy

The functional role of phosphorus-mobilizing bacteria in the rhizosphere of tomato and maize

Phosphorus (P) is an essential plant nutrient. However, global P reserves are being increasingly exploited and surplus P applied by P fertilization is steadily accumulating in the form of plant-unavailable P compounds in arable soils. Future plant production will therefore require a more effective and sustainable P fertilization regime. One promising approach is the use of phosphorus-mobilizing bacteria (PMB), which are able to mobilize P in soil through mineralization or solubilization so effectively that plant P supply is improved. Increases in plant growth and P uptake by the addition of PMB have been reported several times, but PMBs functional mechanisms in soils and plants are still poorly understood. However, an understanding of PMBs functional mechanisms is necessary to evaluate both the potential and limitations of their use as well as to develop practical application recommendations. This thesis aimed to provide a better understanding of PMBs functional mechanisms in soil; the foci here were mechanisms and interactions of P mineralization with indigenous soil microorganisms. We aimed to identify P mineralization-dependent and -independent as well as direct and indirect mechanisms of PMB on soil and plants. To this end, three rhizobox experiments were performed in the greenhouse using tomato and maize as the test plants and Pseudomonas sp. RU47 (RU47) as the PMB. To identify effective P mineralization beyond the level of endogenous microbial activity, a treatment using unselectively cultivated soil bacteria for inoculation was included. Furthermore, the addition of devitalized RU47 cells provided the opportunity to identify indirect mechanisms. In all three rhizobox experiments the activities of acid and alkaline phosphomonoesterases in rhizosphere and bulk soil were determined, as the latter could be clearly identified as being of microbial origin. Effects on microbial community structure in soil were estimated by denaturing gradient gel electrophoresis (DGGE) and/or phospholipid fatty acid analysis. For deeper investigations of potential effects on microbial population composition and possible dependencies on soil conditions, a fourth experiment was performed using maize, three different Pseudomonas strains possessing PMB abilities, and three different soils varying in parameters which included organic C, pH, and P content. Microbiome shifts in soil were quantitatively determined via quantitative PCR using domain- (bacteria, archaea, fungi) and six bacterial phylum-specific primers. Our experiments showed that tomato plants grown under low P availability soil conditions improved in both growth and P uptake when viable RU47 cells were added. This effect was accompanied by increased alkaline phosphatase activity (PA) in the rhizosphere. We also observed plant growth-promotion effects and a trend of increased PA by the addition of dead RU47 cells. Based on DGGE results, which indicated the promotion of indigenous rhizobacteria, we assume a priming effect induced by the addition of C sources in the form of bacterial residues (dead RU47), which resulted in increased indigenous microbial activity in the rhizosphere. In each rhizobox experiment viable RU47 cells were able to colonize the rhizosphere at high abundances, persisting up to 50 days after sowing. We found indications of phytohormonal influences with the addition of both viable and dead RU47 cells, but this was more pronounced in dead than in viable RU47 treatments. Increasing P availability in soil by mineral P fertilization seemed to improve RU47s ability to colonize and persist, which was shown by an increased RU47 abundance in both rhizosphere and bulk soils. However, despite an observable slight tendency, strengthened plant growth-promotion that positively correlated with improved RU47 abundance in the rhizosphere could not be detected. In general, colonization by viable RU47 cells did not significantly affect microbial community structure, either in the rhizosphere or in bulk soil. Using three different PMB strains, including RU47, in three contrasting soils, inoculation effects on the microbial community occurred heterogeneously, differing between the strains, soils, and time. Changes at the domain level were due primarily to nutrient availability in the soil, which differed between the soils and over time. Individual shifts in microbial community structure occurred more frequently in the rhizosphere than in bulk soil, but colonizing PMB neither increased bacterial abundance in rhizosphere bacteria, nor displaced copiotrophic rhizobacteria (indicative of C competition).Phosphor (P) ist ein essentieller Pflanzennährstoff. Die zunehmende Verknappung der globalen P-Ressourcen sowie die steigende P-Anreicherung, in Form von für die Pflanze nicht nutzbaren P-Verbindungen, in agrarwirtschaftlich genutzten Böden zeigen die Dringlichkeit nach einer effizienteren P-Düngung in der zukünftigen Pflanzenproduktion. Einen vielversprechenden Ansatz bieten hierbei Phosphor-mobilisierende Bakterien (PMB). PMB sind in der Lage mittels Mineralisation und Demineralisation P im Boden effektiv zu mobilisieren und für die pflanzliche Aufnahme verfügbar zu machen. Positive Pflanzeneffekte durch den Einsatz von PMB konnten bereits mehrfach nachgewiesen werden; die zugrundeliegenden Funktionsmechanismen blieben jedoch weitgehend unbekannt. Das Wissen um diese Funktionsmechanismen ist jedoch grundlegend, um die Möglichkeiten und Grenzen des praktischen Einsatzes von PMB abschätzen und standortangepasste Anwendungsempfehlungen entwickeln zu können. Ziel dieser Arbeit war es, zu einem besseren Verständnis der Funktionsmechanismen von PMB im Boden beizutragen. Die Schwerpunkte lagen hierbei sowohl auf den Mechanismen der P-Mineralisation als auch auf bodenmikrobielle Interaktionen. Angestrebt wurde die Identifizierung von P-mineralisationsabhängigen und -unabhängigen sowie direkten und indirekten PMB-Mechanismen. Es wurden drei Wurzelkastenversuche unter Verwendung von Tomate und Mais als Testpflanzen und Pseudomonas sp. RU47 (RU47) als PMB-Stamm durchgeführt. Zur Identifizierung einer über die endogene mikrobielle Bodenaktivität hinausgehenden P-Mineralisation wurde jeweils eine Behandlungsgruppe mitgeführt, in der undifferenzierte Bodenbakterien für die Inokulation verwendet wurden. Die Unterscheidung von direkten und indirekten Mechanismen erfolgte durch die Zugabe von abgetöteten RU47-Zellen in einer weiteren Behandlungsgruppe. Es wurde jeweils die saure und alkalische Phosphomonoesteraseaktivität (PA) in Rhizosphäre und Umgebungsboden gemessen, wobei die alkalische PA einem mikrobiellen Ursprung zugewiesen werden kann. Die Untersuchung der Auswirkungen auf die mikrobielle Bodengemeinschaft wurde mittels der denaturierenden Gradienten-Gelelektrophorese (DGGE) und/oder der Phospholipidfettsäuren-Analyse durchgeführt. Für genauere Untersuchungen hinsichtlich möglicher Effekte auf die mikrobielle Bodengemeinschaft sowie deren Beeinflussung durch unterschiedliche Bodeneigenschaften wurde ein viertes Experiment unter Verwendung von Mais durchgeführt. Dieser Versuch enthielt drei Pseudomonas Stämme mit PMB-Eigenschaften sowie drei in ihren Bodeneigenschaften (z.B. C org, P, pH) unterschiedliche Böden. Effekte auf die mikrobielle Bodengemeinschaft wurden quantitativ mittels qPCR unter Verwendung von Domain- (Bakterien, Archaeen, Pilze) und sechs Bakterienphylum-spezifischer Primerpaaren bestimmt. Mit lebenden RU47-Zellen behandelte Tomatenpflanzen zeigten unter P-limitierten Bodenbedingungen eine Verbesserung im Pflanzenwachstum und der P-Aufnahme. Gleichzeitig wurde eine Erhöhung der alkalischen PA in der Rhizosphäre nachgewiesen. Ein pflanzenwachstumsverbessernder Effekt sowie eine erhöhte PA konnte auch bei der Verwendung abgetöteter RU47-Zellen beobachtet werden. An Hand der DGGE-Ergebnisse konnte hier auf eine Förderung endogener Rhizobakterien, vermutlich durch von Zugabe von leicht verfügbarem C in Form von bakteriellen Zellrückständen, geschlossen werden, welche die mikrobielle Aktivität in der Rhizosphäre erhöhte (Priming-Effekt). Lebende RU47-Zellen zeigten in allen Wurzelkastenversuchen eine hohe Rhizosphären-Kompetenz; hoch abundant nachweisbar bis zu 50 Tage nach Aussaat. Es wurden Hinweise auf phytohormonelle Effekte in beiden RU47-Behandlungsgruppen gefunden, diese waren jedoch stärker ausgeprägt in der Gruppe, in der tote RU47-Zellen verwendet wurden. Bei erhöhter P-Verfügbarkeit im Boden (P-Düngung) konnte sowohl in der Rhizosphäre als auch im Umgebungsboden eine erhöhte RU47-Abundanz beobachtet werden. Trotz beobachteter Tendenzen, ein positiv mit der RU47 Rhizosphären-Abundanz korrelierender, pflanzenwachstumsverbessernder Effekt wurde nicht nachgewiesen. Die Besiedlung lebender RU47-Zellen führte weder in der Rhizosphäre noch im Umgebungsboden zu einer Veränderung der mikrobiellen Bodengemeinschaft. Die Verwendung drei verschiedener PMB-Stämme, darunter auch RU47, in drei verschiedenen Böden zeigte einzelne Inokulationseffekte auf die mikrobielle Gemeinschaft, welche jedoch stark zwischen den Stämmen, Böden und der Zeit variierten. Änderungen auf Domainniveau folgten vorwiegend der Nährstoffverfügbarkeit im Boden und variierten somit zwischen den Böden und Probezeitpunkten. Einzelne Inokulationseffekte auf die mikrobielle Bodengemeinschaft wurden häufiger in der Rhizosphäre als im Umgebungsboden beobachtet. Trotz hoher Rhizosphären-Kompetenz wurde weder ein Anstieg der Rhizobakterien-Abundanz noch eine Verdrängung copiotropher Rhizobakterien (C-Konkurrenz)dokumentiert

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

International audienceHepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-argi-nine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phos-phorylation principally suppresses RNA binding whereas one or few strategic dephosphory-lation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target. PLOS Pathogens | https://doi.org/10.1371/journal.ppat

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL

Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide

A SELEX-Screened Aptamer of Human Hepatitis B Virus RNA Encapsidation Signal Suppresses Viral Replication

Background: The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the e RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking e in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. Methodology/Principal Findings: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential e decoys in two large e RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an e decoy that competitively inhibits P protein binding to the authentic e signal on pgRNA. Conclusions/Significance: This study demonstrates the first successful identification of human HBV e aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the abilit

HBV Life Cycle: Entry and Morphogenesis

Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions

The RAB3-RIM Pathway Is Essential for the Release of Neuromodulators

Neurons secrete neuromodulators/neuropeptides from dense-core vesicles (DCVs) by a largely unknown mechanism. Persoon et al. identify RAB3 and RIM1/2 as essential factors. RAB3’s indispensable role is the first distinct feature of DCV secretion as compared to synaptic vesicle secretion

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process

Combining Cell-Free Protein Synthesis and NMR Into a Tool to Study Capsid Assembly Modulation

International audienceModulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures