Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B

Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host–pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N-terminal half of Sec24A and a di-arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross-talk might display a tool to effectively inhibit SVP release

A construção da União Europeia no decorrer de seus tratados instituidores

Resumo: Este estudo apresenta o percurso de construção da União Europeia ao longo dos sucessivos Tratados, demonstrando a formação de suas competências e de seu aparato institucional. Também indica de que maneira o constante conflito entre posição supranacionalista, de um lado, e intergovernamentalista, de outro, implicou num processo de integração nem sempre incontroverso, mas contínuo. Será possível verificar, assim, como a União Europeia tem progressivamente ampliado sua esfera de poder e exigido um número cada vez maior atribuições, em razão das contingências oriundas da vida no bloco. De tal constatação, tornar-se-á mais clara a conclusão de que, hoje, a solução de muitos problemas nacionais e europeus já não pode ser pensada sem uma atuação ao menos conjunta da União Europeia, tendo em vista a profundidade dos vínculos entre os Estados-membros e o grande número de ferramentas titularizadas pelo ente supraestata

Parenteral nutrition support for patients with pancreatic cancer. Results of a phase II study

<p>Abstract</p> <p>Background</p> <p>Cachexia is a common problem in patients (pts) suffering from upper gastrointestinal cancer. In addition, most of these patients suffer from malabsorption and stenosis of the gastrointestinal tract due to their illness. Various methods of supplementary nutrition (enteral, parenteral) are practised. In patients with advanced pancreatic cancer (APC), phase angle, determined by bio-electrical impedance analysis (BIA), seems to be a survival predictor. The positive influence of BIA determinate predictors by additional nutrition is currently under discussion.</p> <p>Methods</p> <p>To examine the impact of additional parenteral nutrition (APN) we assessed outpatients suffering from APC and progressive cachexia. The assessment based on the BIA method. Assessment parameters were phase angle, ECM/BCM index (ratio of extracellular mass to body cell mass), and BMI (body mass index). Patients suffering from progressive weight loss in spite of additional enteral nutritional support were eligible for the study.</p> <p>Results</p> <p>Median treatment duration in 32 pts was 18 [8-35] weeks. Response evaluation showed a benefit in 27 pts (84%) in at least one parameter. 14 pts (43.7%) improved or stabilised in all three parameters. The median ECM/BCM index was 1.7 [1.11-3.14] at start of APN and improved down to 1.5 [1.12-3.36] during therapy. The median BMI increased from 19.7 [14.4-25.9] to 20.5 [15.4-25.0]. The median phase angle improved by 10% from 3.6 [2.3-5.1] to 3.9 [2.2-5.1].</p> <p>Conclusions</p> <p>We demonstrated the positive impact of APN on the assessed parameters, first of all the phase angle, and we observed at least a temporary benefit or stabilisation of the nutritional status in the majority of the investigated patients. Based on these findings we are currently investigating the impact of APN on survival in a larger patient cohort.</p> <p>Trial registration</p> <p>ClinicalTrials.gov Identifier: NCT00919659</p

Impact of ESCRT-II subunits EAP30 and EAP45 on HBV genome maturation.

<p>HuH-7 cells treated with control siRNA (siC) or siRNAs targeting EAP30 or EAP45 were retransfected with the modified pHBVΔHP replicon devoid of foreign promoter elements. <b>A.</b> Lysates were probed with antibodies to EAP30, EAP45, Core, L, and β-actin as indicated. <b>B.</b> Virions released into the cellular supernatants were quantified by real-time PCR of the HBV genomes. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates. <b>C.</b> For quantitative reverse transcription-PCR (qRT-PCR), total mRNAs were isolated, reverse transcribed and used for PCR reactions. To measure HBV pgRNA packaged into intracellular capsids, lysates were subjected to a capsid-specific immunoprecipitation prior to RNA isolation and qRT-PCR. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates.</p

HBV Core interacts with ESCRT-II subunits.

<p>HuH-7 cells were cotransfected with FLAG-tagged EAP20, EAP30, or EAP45 together with pHBV or empty plasmid DNA (pControl) at a 1∶1 DNA ratio. As a control, pHBV was cotransfected with a FLAG-tagged construct derived from Nedd4.1 (HECT). Synthesis of the EAP and HECT constructs is shown by immunoblotting of lysates with anti-FLAG antibodies (left). Numbers to the left of the panel refer to molecular weight standards in kDa. For coimmunoprecipitation (IP), lysates were incubated with anti-core antibodies before Western blotting (WB) with the FLAG-specific antibody (top right). The same blot was stripped and reacted with anti-core antibodies (bottom right). The experiment was repeated three times and a representative image is shown.</p

Impact of ESCRT-II subunits EAP30 and EAP45 on HBV maturation.

<p>HuH-7 cells were treated with control siRNA (siC) or the siRNA duplexes siEAP30#2 or siEAP45#2. After 48 h, cells were retransfected with pHBV, and cell lysates and supernatants were harvested 72 h later. <b>A.</b> Lysates were prepared with detergent and assayed for the synthesis of the HBV core and L envelope (L) proteins by specific Western blotting. <b>B.</b> Lysates were probed for the synthesis of the secretory precore (preC) and S envelope (S) proteins by specific ELISAs. Intracellular amounts of preC and S are expressed as mean units of optical density at 492 nm (<i>n = </i>2) and demonstrated in percent amount relative to siC-treated cells. <b>C.</b> Following treatment with siC, siEAP30#2, or siEAP45#2, cells were transfected with the vector pCore encoding solely the HBV core protein. Cytoplasmic capsids were concentrated by PEG precipitation and separated in a native agarose gel, blotted, and detected with anti-capsid antibodies. <b>D.</b> Cells were treated with the indicated siRNAs followed by transfection with pHBV. Cells were lysed by repetitive freeze-thaw cycles, and intracellular nucleocapsids and enveloped virions were precipitated with capsid- or envelope-specific antibodies, respectively, and assayed by quantitative real time PCR. Error bars indicate the standard deviations from the mean of two experiments measured in duplicates. <b>E.</b> Cells were transfected exactly as outlined in <b>D</b>. For a control, a replication-defective pHBVΔC replicon was included in the Southern blot analysis. Intracellular viral DNA was isolated and processed by Southern blotting using a digoxigenin-dUTP-labeled HBV-specific probe. Numbers to the right refer to marker DNA in kb.</p

Impact of ESCRT-I subunits TSG101 and VPS28 on HBV production.

<p><b>A.</b> HuH-7 cells were treated with control siRNA (siC) or two individual siRNAs targeting TSG101 or VPS28. Cells were subsequently transfected with the pHBV replicon. The knockdown efficacy of the siRNAs was analyzed by Western blotting of cellular lysates using TSG101- or VPS28-specific antibodies. Uniformity of sample loading was confirmed by probing the lysates with anti-β-actin antibodies. Relative protein expression values were determined by densitometric analysis and demonstrated in percent amount relative to control cells. To probe for cytotoxicity, cellular supernatants were assayed for LDH activity and indicated as factors of increase relative to control cells. <b>B.</b> The number of virions released into the cell culture supernatants were quantitated by real-time PCR of the viral genomes and demonstrated in percent amount relative to control siRNA-treated cells. Error bars indicate the standard deviations from the mean of four experiments measured in duplicates. <b>C.</b> Co-depletion effects of ESCRT-I-specific siRNAs. Control siRNA or the two siRNAs targeting either TSG101 or VPS28 were introduced into HuH-7 cells prior to transfection with pHBV. Cell lysates were prepared and subjected to Western blotting using anti-TSG101, anti-VPS28, and anti-ß-actin antibodies. TSG101- and VPS28-specific band intensities were quantitated and demonstrated in percent amount relative to siC-transfected cells.</p

HBV Core colocalizes with ESCRT-II subunits.

<p><b>A.</b> HuH-7 cells were transfected with FLAG-tagged EAP20, EAP30, or EAP45 and immunostained with mouse anti-FLAG and rabbit anti-CD63 antibodies. After staining with secondary antibodies, cells were analyzed by deconvolution fluorescence microscopy. The staining pattern of the EAP constructs is shown in green, and the fluorescent signal of CD63 is in red. The overlays of the fluorescence patterns are shown in the right column with yellow color indicating colocalization. Insets in these images display enlarged sections that are shown in the very right column. DNA staining is shown in blue. Bar, 10 µm. <b>B.</b> Cells were transfected with pHBV alone or FLAG-tagged EAP20, EAP30, or EAP45 and costained with rabbit anti-core (red) and mouse anti-FLAG (green) antibodies. Colocalization between ESCRT-II subunits and core is indicated in yellow in the right column. Outlined areas of these images are shown at larger magnification. DNA staining is shown in blue. Bar, 10 µm. The degree of colocalization was estimated using the Axiovision image analysis software (Zeiss). Ten cells from three independent transfection experiments were quantitated for the degree of overlap (Cell) or spatial pattern of overlap by analyzing five regions of interest (ROI) per cell. The average of colocalization is plotted in the right graphs.</p

Impact of ESCRT-II subunits EAP20, EAP30, and EAP45 on HBV production.

<p><b>A.</b> HuH-7 cells were treated with control siRNA (siC), a siRNA pool targeting EAP20, or two different siRNA duplexes directed against EAP30 or EAP45. After 48 h, cells were retransfected with pHBV, and lysates and supernatants were harvested 72 h later. To probe for the efficiency of the knockdowns, lysates were immunoblotted with antibodies against EAP20, EAP30, and EAP45. Identical sample loading was assessed by anti-β-actin Western blotting, and relative protein expression values were determined by densitometric analysis and demonstrated in percent amount relative to control cells. To probe for cell lysis, supernatants were assayed for LDH activity. <b>B.</b> Virions released into the cellular supernatants were quantified by real-time PCR of the HBV genomes. Error bars indicate the standard deviations from the mean of four experiments measured in duplicates. <b>C.</b> Kinetic and co-depletion effects of the ESCRT-II-specific siRNAs. Cells were treated with the indicated siRNAs for 48 h, transfected with pHBV and lysed after 24, 48, or 72 h DNA transfection. RNAi effects on the expression of EAP20, EAP30, and EAP45 were analyzed by specific immunoblotting. A non-specific band stained by the antisera served as a control for identical gel loading (LC).</p

Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B

Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host–pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N-terminal half of Sec24A and a di-arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross-talk might display a tool to effectively inhibit SVP release