The Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic agent responsible for severe hemorrhagic fever in humans. Understanding the molecular mechanisms governing its replication is critical for developing effective antiviral strategies. VP35-TurboID immunosuppression coupled with quantitative mass spectrometry identified Septin9, the host GTP-binding protein which played a role in cytoskeletal regulation, as a novel interactor of VP35. Western blotting and Far-Western blotting confirmed the direct interaction and demonstrated that the C-terminal region of VP35 was the critical binding domain. Functionally, EBOV replication as well as the formation of viral inclusion bodies (VIBs) was demonstrated to be significantly suppressed by Septin9 knockdown and depletion, as shown by the EBOV minigenome (EBOV MG) and the transcription- and replication-competent virus-like particles (trVLPs) system. This study reveals that VP35 engages in a specific interaction with the GTP-binding protein Septin9, thereby impeding EBOV replication through the disruption of inclusion bodies. The overarching objective of this study is to significantly enhance our understanding about the pathogenic mechanism of EBOV and offer a robust theoretical foundation and solid empirical support for the formulation of innovative therapeutic strategies against EBOV.
Virus Replication/physiology* ; Septins/physiology* ; Humans ; Ebolavirus/physiology* ; Inclusion Bodies, Viral/metabolism* ; Viral Regulatory and Accessory Proteins/metabolism* ; Hemorrhagic Fever, Ebola/virology*

Nuclear bodies are membrane-free nuclear substructures that are localized in the mammalian nuclear matrix region. They are multiprotein complexes that recruit other proteins to participate in various cellular activities, such as transcription, RNA splicing, epigenetic regulation, tumorigenesis and antiviral defense. It is of great significance to clarify the functions and regulatory mechanisms of nuclear bodies to probe related diseases and virus-host interactions. This review takes several nuclear bodies associated proteins as examples, summarizes the formation process, structure and functions of nuclear bodies, and focuses on their important roles in antiviral infection. It is expected to provide new insight into host antiviral mechanisms.
Animals ; Cell Nucleus ; Epigenesis, Genetic ; Intranuclear Inclusion Bodies/metabolism* ; Nuclear Proteins/metabolism*

Primary age-related tauopathy (PART) is characterized by tau neurofibrillary tangles (NFTs) in the absence of amyloid plaque pathology. In the present study, we analyzed the distribution patterns of phosphorylated 43-kDa TAR DNA-binding protein (pTDP-43) in the brains of patients with PART. Immunohistochemistry and immunofluorescence double-labeling in multiple brain regions was performed on brain tissues from PART, Alzheimer's disease (AD), and aging control cases. We examined the regional distribution patterns of pTDP-43 intraneuronal inclusions in PART with Braak NFT stages > 0 and ≤ IV, and a Thal phase of 0 (no beta-amyloid present). We found four stages which indicated potentially sequential dissemination of pTDP-43 in PART. Stage I was characterized by the presence of pTDP-43 lesions in the amygdala, stage II by such lesions in the hippocampus, stage III by spread of pTDP-43 to the neocortex, and stage IV by pTDP-43 lesions in the putamen, pallidum, and insular cortex. In general, the distribution pattern of pTDP-43 pathology in PART cases was similar to the early TDP-43 stages reported in AD, but tended to be more restricted to the limbic system. However, there were some differences in the distribution patterns of pTDP-43 between PART and AD, especially in the dentate gyrus of the hippocampus. Positive correlations were found in PART between the Braak NFT stage and the pTDP-43 stage and density.
Aged ; Aged, 80 and over ; Aging ; metabolism ; pathology ; Brain ; metabolism ; pathology ; DNA-Binding Proteins ; metabolism ; Disease Progression ; Female ; Humans ; Immunohistochemistry ; Inclusion Bodies ; pathology ; Male ; Middle Aged ; Neurofibrillary Tangles ; metabolism ; pathology ; Neurons ; metabolism ; pathology ; Severity of Illness Index ; Tauopathies ; metabolism ; pathology

Viral infection induces numerous tripartite motif (TRIM) proteins to control antiviral immune signaling and viral replication. Particularly, SPRY-containing TRIM proteins are found only in vertebrates and they control target protein degradation by their RING-finger and SPRY domains, and proper cytoplasmic localization. To understand TRIM30 function, we analyzed its localization pattern and putative roles of its RING-finger and SPRY domains. We found that TRIM30 is located in actin-mediated cytoplasmic bodies and produces colocalized ubiquitin chains in SPRY domain- and RING-finger domain-dependent ways that are degraded by autophagy and the proteasome. These results suggest a TRIM protein-dependent degradation mechanism by cytoplasmic body formation with actin networks.
Amino Acid Sequence ; Animals ; Autophagy ; Cell Line ; Inclusion Bodies/*metabolism ; Intracellular Signaling Peptides and Proteins/chemistry/genetics/*metabolism ; Mice ; Molecular Sequence Data ; Polyubiquitin/*metabolism ; Proteasome Endopeptidase Complex/metabolism ; Protein Interaction Domains and Motifs ; Protein Transport ; Proteolysis ; RING Finger Domains

OBJECTIVETo investigate histopathology and proteinopathy in the spinal cord of patients with common neurodegenerative diseases.

METHODSSpinal cord tissues from clinically and neuropathologically confirmed neruodegnerative diseases were enrolled in this study, including 3 cases of multiple system strophy, 4 cases of amyotrophic lateral sclerosis, 5 cases of Alzheimer's disease (AD, included 2 cases of AD combined with Parkinson's disease), 2 cases of progressive supranuclear palsy, 1 case of dementia with lewy body and 1 case of corticobasal degeneration from 1955 to 2013 at Chinese People's Liberation Army General Hospital. Four normal control cases were also included. Routine HE and Gallyas-Braak staining, and immunohistochemical stainings for anti-PHF tau (AT8), anti-α-synuclein, anti-TDP-43 and anti-ubiquitin were performed.

RESULTSExamination of the spinal cord in 3 cases with multiple system strophy revealed severe neuron loss in the intermediolateral nucleus of thoracic segment and Onuf's nucleus of the sacral segment, along with moderate neuron loss in the anterior horn of the cervical segment and mild myelin pallor in the anterior funiculus and anterolateral funiculus in the cervical and thoracic segments. Large amount of argentophilic, ubiquitin and synuclein positive oligodendroglial cytoplasmic inclusions were found widely distributed in the anterior horn and the anterior funiculus and anterolateral funiculus of the full spinal cord. Severe neuron loss and several morphological changes with gliosis in the anterior horn and severe loss of myelin in the anterior funiculus and anterolateral funiculus of the full spinal cord were observed in 4 cases of amyotrophic lateral sclerosis, 2 of which were found with Bunina bodies in neurons of the anterior horn. Three amyotrophic lateral sclerosis cases had ubiquitin-positive neuronal inclusions and TDP-43 positive neuronal and glial inclusions in the anterior horn at cervical and lumbar segments. A few argentophilic, tau positive neurofibrillary tangles (NFTs) and neuropil threads in the anterior horn at cervical and lumbar segments were found in 4 AD cases. Examination of spinal cord in 2 cases with Parkinson's disease combined with AD and 1 case with dementia with lewy body revealed severe neuron loss in the intermediolateral nucleus of thoracic segment, and a few synuclein positive lewy bodies and neuritis were also observed. There was mild neuron loss in the anterior horn at cervical and lumbar segments, along with some argentophilic, tau positive globous NFTs and many argentophilic, tau positive neutrophil threads were observed in 2 progressive supranuclear palsy cases and 1 corticobasal degeneration case.

CONCLUSIONEach common neurodegenerative diseases of the spinal cord including multiple system strophy, amyotrophic lateral sclerosis and Parkinson's disease has its own specific histopathology and proteinopathy characteristics.

Alzheimer Disease ; pathology ; Amyotrophic Lateral Sclerosis ; pathology ; DNA-Binding Proteins ; metabolism ; Humans ; Immunohistochemistry ; Inclusion Bodies ; pathology ; Neurodegenerative Diseases ; pathology ; Neurofibrillary Tangles ; pathology ; Neurons ; pathology ; Parkinson Disease ; pathology ; Spinal Cord ; pathology ; Ubiquitin ; metabolism ; alpha-Synuclein ; metabolism

Fibroblast growth factor -21 (FGF-21) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.
Animals ; Bacteria ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; Disease Models, Animal ; Fibroblast Growth Factors ; biosynthesis ; Genetic Vectors ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; isolation & purification ; Inclusion Bodies ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis

This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.
Adenocarcinoma ; metabolism ; pathology ; Aminoglycosides ; chemistry ; metabolism ; Antibiotics, Antineoplastic ; chemistry ; metabolism ; Apoproteins ; chemistry ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Collagenases ; immunology ; Enediynes ; chemistry ; metabolism ; Escherichia coli ; chemistry ; metabolism ; Humans ; Inclusion Bodies ; chemistry ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Lung Neoplasms ; metabolism ; pathology ; Protein Binding ; Recombinant Fusion Proteins ; chemistry ; metabolism ; Single-Chain Antibodies ; chemistry ; metabolism

To investigate the components of fibrillous inclusion body (FIB), which was formed in packaging cells during the replication of human adenovirus type 41 (Ad41), Ad41 long fiber knob (LFK) and short fiber knob (SFK) proteins were expressed in prokaryote respectively and then used to immunize BALI mice for preparation of anti-LFK serum and anti-SFK sera. The activity and specificity of anti-LFK and an ti-SFK sera were confirmed with Western blot, indirect immunofluorescence assay (IFA) and immunonegative staining, suggesting these sera could be applied in immuno-colloidal gold labelling electron microscopy (EM). 293TE cells were infected with wild-type Ad41. Ultrathin sections of infected cells were made, and labelled with immuno-colloidal gold technique using anti-Ad41 sera, anti-LFK sera, anti-SFK sera, or anti-fiber monoclonal antibody 4D2, respectively. The labelled sections were observed under EM, and the results demonstrated that both Ad41 long fiber protein and short fiber protein were included FIB.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; metabolism ; ultrastructure ; Animals ; Female ; Humans ; Inclusion Bodies, Viral ; ultrastructure ; Mice ; Mice, Inbred BALB C ; Microscopy, Immunoelectron

Calcium-Binding Proteins ; metabolism ; Diagnosis, Differential ; Female ; Fibroma ; metabolism ; pathology ; surgery ; Humans ; Inclusion Bodies ; pathology ; Infant ; Microfilament Proteins ; metabolism ; Skin Neoplasms ; metabolism ; pathology ; surgery ; Soft Tissue Neoplasms ; pathology ; Tendons ; pathology ; Toes ; Vimentin ; metabolism

OBJECTIVETo investigate the effect of Yisui Shengxue Granule (, YSSXG), a complex Chinese medicine, on the oxidative damage of erythrocytes from patients with hemoglobin H (HbH) disease.

METHODSTwenty-two patients with HbH disease and 22 healthy volunteers were observed. YSSXG was given to patients with HbH disease for 3 months. Before and after the 3-month treatment, blood parameters [hemoglobin (Hb), red blood cells (RBCs), and reticulocyte percent (Ret)] were examined; inclusion bodies in erythrocytes were observed by transmission electron microscopy (TEM); activities of antioxidant defense enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (Cat)] and erythrocyte membrane malondialdehyde (MDA) concentrations were determined.

RESULTSIn patients with HbH disease, measured values of RBC and Hb obtained from the first to the third months after treatment with YSSXG were significantly higher than before treatment (P<0.01). Measured values of Ret from the second to the third months after treatment were significantly lower than before treatment (P<0.05 and P<0.01, respectively). Prior to treatment with YSSXG, TEM images of RBCs showed the presence of numerous inclusion bodies. After treatment with YSSXG, the amount and volume of inclusion bodies decreased. Treatment with YSSXG also led to a significant increase in SOD activity (P<0.01), a decrease in Cat activity (P<0.01), and no significant differences in GSHPx activity (P>0.05) or MDA concentration (P>0.05). However, compared with the healthy counterparts, SOD, GSH-Px, and Cat activities presented at high levels (P<0.01) both before and after treatment.

CONCLUSIONSYSSXG could improve the degree of hemolysis and anemia in patients with HbH disease. The mechanism may be related to its antioxidative effects, which could elevate the activity of total SOD in erythrocytes and efficiently inhibit the oxidative precipitation of β-globin chains.

Adolescent ; Adult ; Catalase ; metabolism ; Child ; Child, Preschool ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Erythrocyte Membrane ; drug effects ; metabolism ; ultrastructure ; Erythrocytes ; drug effects ; enzymology ; pathology ; ultrastructure ; Female ; Glutathione Peroxidase ; metabolism ; Humans ; Inclusion Bodies ; drug effects ; ultrastructure ; Male ; Malondialdehyde ; metabolism ; Oxidative Stress ; drug effects ; Superoxide Dismutase ; metabolism ; Young Adult ; alpha-Thalassemia ; blood ; drug therapy ; pathology