Biopsies from patients show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, ...cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.
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•Identification of the binding partner mediating cytoplasmic transport of HBV capsids.•Transport was restricted to mature and empty capsids.•The domains on capsid and dynein LL1 were ...identified.•Transport inhibition may block infection and increase elimination of infected hepatocytes.
Hepatitis B virus (HBV) has a DNA genome but replicates within the nucleus by reverse transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are a major target for T cell-mediated immune responses. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal.
We used virion-derived capsids containing mature rcDNA (matC) and empty capsids (empC). RNA-containing capsids (rnaC) were used as a control. The investigations comprised pull-down assays for identification of cellular interaction partners, immune fluorescence microscopy for their colocalization and electron microscopy after microinjection to determine their biological significance.
matC and empC underwent active transport through the cytoplasm towards the nucleus, while rnaC was poorly transported. We identified the dynein light chain LL1 as a functional interaction partner linking capsids to the dynein motor complex and showed that there is no compensatory transport pathway. Using capsid and dynein LL1 mutants we characterized the required domains on the capsid and LL1.
This is the first investigation on the detailed molecular mechanism of how matC pass the cytoplasm upon infection and how empC can be actively removed from the cytoplasm into the nucleus. Considering that hepatocytes with cytoplasmic capsids are better recognized by the T cells, we hypothesize that targeting capsid DynLL1-interaction will not only block HBV infection but also stimulate elimination of infected cells.
In this study, we identified the molecular details of HBV translocation through the cytoplasm. Our evidence offers a new drug target which could not only inhibit infection but also stimulate immune clearance of HBV infected cells.
The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences ...in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin-cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin alpha, importin beta, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.
Background
Telerehabilitation could benefit a large population by increasing adherence to rehabilitation protocols.
Objective
Our objective was to review and discuss the use of cost-utility ...approaches in economic evaluations of telerehabilitation interventions.
Methods
A review of the literature on PubMed, Scopus, Centres for Review and Dissemination databases (including the HTA database, the Database of Abstracts of Reviews of Effects, and the NHS Economic Evaluation Database), Cochrane Library, and ClinicalTrials.gov (last search on February 8, 2021) was conducted in accordance with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The inclusion criteria were defined in accordance with the PICOS (population, intervention, comparison, outcomes, and study design) system: the included studies had to evaluate patients in rehabilitation therapy for all diseases and disorders (population) through exercise-based telerehabilitation (intervention) and had to have a control group that received face-to-face rehabilitation (comparison), and these studies had to evaluate effectiveness through gain in quality of life (outcome) and used the design of randomized and controlled clinical studies (study).
Results
We included 11 economic evaluations, of which 6 concerned cardiovascular diseases. Several types of interventions were assessed as telerehabilitation, consisting in monitoring of rehabilitation at home (monitored by physicians) or a rehabilitation program with exercise and an educational intervention at home alone. All studies were based on randomized clinical trials and used a validated health-related quality of life instrument to describe patients’ health states. Four evaluations used the EQ-5D, 1 used the EQ-5D-5L, 2 used the EQ-5D-3L, 3 used the Short-Form Six-Dimension questionnaire, and 1 used the 36-item Short Form survey. The mean quality-adjusted life years gained using telerehabilitation services varied from –0.09 to 0.89. These results were reported in terms of the probability that the intervention was cost-effective at different thresholds for willingness-to-pay values. Most studies showed results about telerehabilitation as dominant (ie, more effective and less costly) together with superiority or noninferiority in outcomes.
Conclusions
There is evidence to support telerehabilitation as a cost-effective intervention for a large population among different disease areas. There is a need for conducting cost-effectiveness studies in countries because the available evidence has limited generalizability in such countries.
Trial Registration
PROSPERO CRD42021248785; https://tinyurl.com/4xurdvwf
Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct ...steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Cα, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.
In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the ...nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.
AdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.
Every year, one million people dies from Hepatitis B virus. As for other viruses, its genetic material is enclosed by a capsid whose segmentation and classification is essential. In this paper, we ...present a novel capsid segmentation technique which is a combination of a "à trous" wavelet process (for background filtering) and a graph-based structure (for segmentation and classification). Capsids were acquired in transmission electron microscopy (TEM) as a set of 9 series of 40 images each. Our technique achieved as much as 80% of capsid detection and classification for 8 of the series, reaching more than 90% for 5 of them.
Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in ...the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin α2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin α2, not α1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin α2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein—karyopherin α2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin α2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin α2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin α2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin α2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.
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