The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major ...insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.
Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary ...for particle infectivity in related viruses Harada, T. et al., (2000) J. Virol. 74, 9498–9506. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza Hay, A.J. et al. (1985) EMBO J. 4, 3021–3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485–489 and has recently been shown to be active in combination with current HCV therapies.
The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride ...compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.
1. Plant invasions are predicted to accelerate in a world with increased anthropogenic disturbance. Non-native species pre-adapted to these disturbances may especially be poised to invade novel ...communities. Conservation managers therefore need predictions of how to alter disturbances to maximize the persistence of native biodiversity. 2. We tested a multivariate hypothesis about the causal mechanisms underlying plant invasions in an ephemeral wetland in South Island, New Zealand, to inform management of this biodiverse but globally imperilled habitat. Our approach details among the first applications in ecology of Bayesian structural equation modelling, demonstrating its potential to inform management by disentangling the relative importance of strongly intercorrelated processes. 3. We found that invasion by non-native plants was lowest in sites where the physical disturbance caused by flooding was both intense and frequent. This effect was stronger than the positive response of non-native species to high soil N supply, which was positively related to flooding. 4. Sites flooded over a 4-year period had greater reductions in invasion than those associated with floods in the year prior to plot measurement because non-native species lacked traits for long-term persistence beneath water. Grazer exclusion had a small positive effect on invasion, as non-native species were preferentially selected by the herbivores at our site. 5. Our results show that only species adapted to the dominant disturbance regimes at a site may become successful invaders. Species native to ephemeral wetlands have specially evolved traits that allow them to persist and dominate in these sites. 6. Synthesis and applications. Predictions of invasions in a world of multiple disturbances clearly need to consider whether the evolutionary history of non-native species predisposes them to invade novel communities. Maintaining hydrological and nutrient regimes of ephemeral wetlands will limit the number of introduced species that are pre-adapted to become invasive.
Abstract
With the growing availability of data within various scientific domains, generative models hold enormous potential to accelerate scientific discovery. They harness powerful representations ...learned from datasets to speed up the formulation of novel hypotheses with the potential to impact material discovery broadly. We present the Generative Toolkit for Scientific Discovery (GT4SD). This extensible open-source library enables scientists, developers, and researchers to train and use state-of-the-art generative models to accelerate scientific discovery focused on organic material design.
We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. ...Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.
Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane ...channel activity
in vitro and is essential for replication
in vivo though its precise role in the virus life cycle is unknown
. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient
in vitro assay for exploiting p7 as a therapeutic target.
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