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Veterinary Research Aug 2021This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/β-catenin signalling pathway and its effects on...
Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/β-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells.
This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/β-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of β-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/β-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the β-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/β-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.
Topics: Animals; Apoptosis; Avian Leukosis; Avian Leukosis Virus; Avian Proteins; Carcinogenesis; Cell Line; Cell Movement; Chickens; Poultry Diseases; Telomerase; Virus Replication; Wnt Signaling Pathway
PubMed: 34412690
DOI: 10.1186/s13567-021-00979-x -
Poultry Science Oct 2021Avian leukemia is a common malignant disease, and and its regulatory mechanism is complex. As the most extensive tumor suppressor gene in cancer research, p53 can...
Avian leukemia is a common malignant disease, and and its regulatory mechanism is complex. As the most extensive tumor suppressor gene in cancer research, p53 can control multiple functions such as that of DNA repair, induction of apoptosis, cell cycle arrest and so on. In view of the diversity associated with varied function of p53, this study analyzed the possible effect of gene on ALV-J replication and its regulatory mechanism. We successfully constructed a p53 knockout DF-1 cell line (p53-KO-DF-1 cells) by using CRISPR-Cas9 system. When ALV-J was co-infected with DF-1 and p53-KO-DF-1 cells, it was found that compared with wild-type DF-1 cells, the viral copy number of p53-KO-DF-1 cells infected with ALV-J increased significantly 48 h after infection, whereas the expression of innate immune factors such as Il-2,TNF- α, IFN- γ and MX1 decreased significantly. Detection of p53-related tumor genes indicated that after p53 deletion, the expression of c-myc, bcl-2, and bak increased significantly, while the expression of p21 and p27 was noted to be decreased. The cell cycle distribution and apoptosis of the 2 cell lines was detected by flow cytometry analysis. The results showed that p53 knockout prevented G0/G1 and G2 M phase arrest induced by ALV-J, and substantially decreased the rate of apoptosis. Overall, the results indicated that p53 gene can effectively inhibits ALV-J replication by regulating important cellular processes, and p53 gene related proteins involved in cell cycle activity may function as the key targets for the prevention and treatment of ALV-J.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Cell Line; Chickens; Tumor Suppressor Protein p53
PubMed: 34411963
DOI: 10.1016/j.psj.2021.101374 -
The Journal of Physical Chemistry... Aug 2021During the maturation step, the retroviral capsid proteins (CAs) assemble into polymorphic capsids. Their acute curvature is largely determined by 12 pentamers inserted...
During the maturation step, the retroviral capsid proteins (CAs) assemble into polymorphic capsids. Their acute curvature is largely determined by 12 pentamers inserted into the hexameric lattice. However, how the CA switches its conformation to control assembly curvature remains unclear. We report the high-resolution structural model of the Rous sarcoma virus (RSV) CA = 1 capsid, established by molecular dynamics simulations combining solid-state NMR and prior cryoelectron tomography restraints. Comparing this with our previous model of the RSV CA tubular assembly, we identify the key residues for dictating the incorporation of acute curvatures. These residues undergo large torsion angle changes, resulting in a 34° rotation of the C-terminal domain relative to its N-terminal domain around the flexible interdomain linker, without substantial changes of either the conformation of individual domains or the assembly contact interfaces. This knowledge provides new insights to help decipher the mechanism of the retroviral capsid assembly.
Topics: Capsid; Capsid Proteins; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Pliability; Protein Conformation; Protein Domains; Rous sarcoma virus
PubMed: 34374542
DOI: 10.1021/acs.jpclett.1c01769 -
Viruses Jul 2021Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector...
Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123 AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123 AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.
Topics: Aged; Alpharetrovirus; Animals; Cell Line, Tumor; Female; Genetic Therapy; Genetic Vectors; Humans; Immunotherapy, Adoptive; Interleukin-3 Receptor alpha Subunit; Killer Cells, Natural; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Primary Cell Culture; Receptors, Chimeric Antigen; Transduction, Genetic; Transgenes; Xenograft Model Antitumor Assays
PubMed: 34372571
DOI: 10.3390/v13071365 -
Journal of Virology Sep 2021During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. "Complex"...
During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. "Complex" retroviruses, such as human immunodeficiency virus (HIV), use -acting elements on the unspliced gRNA in conjunction with -acting viral proteins to facilitate this escape. "Simple" retroviruses, such as Mason-Pfizer monkey virus (MPMV) and murine leukemia virus (MLV), exclusively use -acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous sarcoma virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling-deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to wild-type (WT) virus and surprisingly is replication competent, albeit with a slower rate of spread than observed in WT virus. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. Rous sarcoma virus (RSV) Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to - and -acting mechanisms for gRNA export.
Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Cell Nucleus; Gene Products, gag; Genome, Viral; Humans; Mice; RNA, Viral; Retroviridae; Rous sarcoma virus; Virion; Virus Assembly
PubMed: 34319154
DOI: 10.1128/JVI.00648-21 -
Virulence Dec 2021Avian leukosis virus subgroup J (ALV-J) generally induces hemangioma, myeloid leukosis, and immunosuppression in chickens, causing significant poultry industry economic...
Avian leukosis virus subgroup J (ALV-J) generally induces hemangioma, myeloid leukosis, and immunosuppression in chickens, causing significant poultry industry economic losses worldwide. The unusual gene of ALV-J, with low homology to other subgroups of ALVs, is associated with its unique pathogenesis. However, the exact molecular basis for the pathogenesis and oncogenesis of ALV-J is still not fully understood. In this study, ALV-J infection and the overexpression of Env could efficiently downregulate the phosphorylation of SHP-2 (pSHP-2) and . The membrane-spanning domain (MSD) in Env Gp37 was the functional domain responsible for pSHP-2 downregulation. Moreover, the overexpression of SHP-2 could effectively promote the replication of ALV-J, whereas knockout or allosteric inhibition of SHP-2 could inhibit ALV-J replication. In addition, the knockout of endogenous chicken SHP-2 could significantly increase the proliferation ability of DF-1 cells. All these data demonstrate that SHP-2 dephosphorylated by ALV-J Env could efficiently promote ALV-J replication, highlighting the important role of SHP-2 in the pathogenesis of ALV-J and providing a new target for developing antiviral drugs against ALV-J.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; Genes, env; Poultry Diseases; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Virus Replication
PubMed: 34167452
DOI: 10.1080/21505594.2021.1939952 -
Poultry Science Jul 2021Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused huge economic losses in the poultry industry due to its great pathogenicity and...
Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused huge economic losses in the poultry industry due to its great pathogenicity and transmission ability. However, the continuous emergence of new strains would bring challenges to diagnosis and control of ALV-J. .This study focuses on preparing the monoclonal antibody (MAb) against ALV-J Gp85 and identifying its epitope. The truncated ALV-J gp85 gene fragment was amplified and then cloned into expression vectors. Purified GST-Gp85 was used to immune mice and His-Gp85 was used to screen MAb. Finally, a hybridoma cell line named J16 that produced specific MAb against the ALV-J. Immunofluorescence assay showed that MAb J16 specifically recognized ALV-J rather than ALV-A or ALV-K infected DF-1 cells. To identify the epitope recognized by MAb J16, fourteen partially overlapping ALV-J Gp85 fragments were prepared and tested by Western blot. The results indicated that peptide 150-LIRPYVNQ-157 was the minimal epitope of ALV-J Gp85 recognized by MAb J16. Alignment analysis of Gp85 from different ALV subgroups showed that the epitope keep high conservation among 36 ALV-J strains, but significant different from that of ALV subgroup A, B, C, D, E and K. Overall, we prepared a MAb specific against ALV-J and identified peptide 150-LIRPYVNQ-157 as a novel specific epitope of ALV-J Gp85, which may assist in laying the foundation for specific ALV-J detection methods.
Topics: Animals; Antibodies, Monoclonal; Avian Leukosis; Avian Leukosis Virus; Chickens; Epitopes; Mice; Poultry Diseases; Viral Envelope Proteins
PubMed: 34116348
DOI: 10.1016/j.psj.2021.101108 -
Poultry Science Jul 2021The avian leukosis virus (ALV) strain DL00766 was isolated from a farm in China. The phylogenetic analysis showed that env had the highest homology with the E subgroup...
The avian leukosis virus (ALV) strain DL00766 was isolated from a farm in China. The phylogenetic analysis showed that env had the highest homology with the E subgroup reference strain, ranging from 94.5% to 94.9%, whereas gp85 had the highest homology with the B and E subgroups, which were 89.0% to 91.3% and 91.3% to 91.8%. In addition, point mutation analysis of gp85 showed that a 400 bp long fragment in gp85 of DL00766 had the highest homology with subgroup B, ranging from 90.1% to 97.5%, and only 82.7% to 83.1% with E subgroup. These results indicate, DL00766 may be an AVL subgroup E isolate with a subgroup B-like gp85 region. This is also the first finding that the E subgroup is used as a recombinant subject, and the subgroup B provides a recombinant virus of an exogenous gene.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; China; Phylogeny; Poultry Diseases; Viral Envelope Proteins
PubMed: 34111609
DOI: 10.1016/j.psj.2021.101137 -
Molecules (Basel, Switzerland) May 2021Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus which has caused heavy losses to the poultry breeding industry. Currently, there is no effective...
Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus which has caused heavy losses to the poultry breeding industry. Currently, there is no effective medicine to treat this virus. In our previous experiments, the low-molecular-weight polysaccharide (SFP) was proven to possess antiviral activity against ALV-J, but its function was limited to the virus adsorption stage. In order to improve the antiviral activity of the SFP, in this study, three new SFP long-chain alkyl group nanomicelles (SFP-C12M, SFP-C14M and SFP-C16M) were prepared. The nanomicelles were characterized according to their physical and chemical properties. The nanomicelles were characterized by particle size, zeta potential, polydispersity index, critical micelle concentration and morphology. The results showed the particle sizes of the three nanomicelles were all approximately 200 nm and SFP-C14M and SFP-C16M were more stable than SFP-C12M. The newly prepared nanomicelles exhibited a better anti-ALV-J activity than the SFP, with SFP-C16M exhibiting the best antiviral effects in both the virus adsorption stage and the replication stage. The results of the giant unilamellar vesicle exposure experiment demonstrated that the new virucidal effect of the nanomicelles might be caused by damage to the phospholipid membrane of ALV-J. This study provides a potential idea for ALV-J prevention and development of other antiviral drugs.
Topics: Adsorption; Animals; Antiviral Agents; Avian Leukosis Virus; Chemistry, Pharmaceutical; Chickens; Dietary Carbohydrates; Gene Expression Regulation; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Light; Magnetic Resonance Spectroscopy; Micelles; Microscopy, Electron, Scanning; Nanoparticles; Nanotechnology; Particle Size; Polysaccharides; Poultry; Poultry Diseases; Sargassum; Scattering, Radiation; Spectroscopy, Fourier Transform Infrared
PubMed: 34071584
DOI: 10.3390/molecules26113265 -
Viruses May 2021Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and...
Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.
Topics: Cell Membrane; Cell Nucleus; Cells, Cultured; Cytoplasm; Dimerization; Genome, Viral; Humans; In Situ Hybridization, Fluorescence; Molecular Imaging; RNA, Viral; Rous sarcoma virus
PubMed: 34068261
DOI: 10.3390/v13050903