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MBio Apr 2020Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA...
Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944-3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790-6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.
Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Cell Line, Transformed; Cell Nucleus; Fibroblasts; Gene Products, gag; Genome, Viral; Microscopy, Confocal; Quail; RNA, Viral; Rous sarcoma virus; Time-Lapse Imaging; Virus Assembly
PubMed: 32265329
DOI: 10.1128/mBio.00524-20 -
Journal of Virology Oct 2015Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma...
UNLABELLED
Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.
IMPORTANCE
Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.
Topics: Cell Membrane; Cholesterol; Escherichia coli; Gene Expression; Gene Products, gag; HIV-1; Hydrodynamics; Lipid Bilayers; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol 4,5-Diphosphate; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Rous sarcoma virus; Virion
PubMed: 26246573
DOI: 10.1128/JVI.01628-15 -
Poultry Science May 2018In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial...
In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.
Topics: Adenoviridae Infections; Animals; Avian Leukosis; Avian Leukosis Virus; Chicken anemia virus; Chickens; Circoviridae Infections; Coinfection; Female; Fowl adenovirus A; Phylogeny; Poultry Diseases; Reticuloendotheliosis virus; Retroviridae Infections; Tumor Virus Infections
PubMed: 29509913
DOI: 10.3382/ps/pex414 -
Proceedings of the National Academy of... Apr 2016This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This...
This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This insight was made possible by in-depth study of RSV-transformed rat XC cells, which were called virogenic because they indefinitely carry virus genetic information in the absence of any infectious virus production. However, the virus was rescued by association of XC cells with chicken fibroblasts, allowing cell fusion between both partners. This and additional studies led to the interpretation that the RSV genome gets integrated into the host cell genome as a provirus. Study of additional rodent virogenic cell lines provided evidence that the transcript of oncogene v-src can be transmitted to other retroviruses and produce cell transformation by itself. As discussed in the text, two main questions related to nonpermissiveness to retrovirus infection remain to be solved. The first is changes in the retrovirus envelope gene allowing virus entry into a nonpermissive cell. The second is the nature of the permissive cell functions required by the nonpermissive cell to ensure infectious virus production. Both lines of investigation are being pursued.
Topics: Animals; Cell Fusion; Cell Line; Cell Transformation, Viral; Chickens; Gene Products, env; Genome, Viral; Oncogene Protein pp60(v-src); Proviruses; Rats; Rous sarcoma virus
PubMed: 27035994
DOI: 10.1073/pnas.1602317113 -
Viruses Dec 2023Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was...
Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of primers were designed, based on the conserved region of the gp85 gene. An exploration of the optimal primer concentration and annealing temperature were carried out, for better performance of the nanoPCR assay. According to the results, the multiple nanoPCR assay amplified 336 pb, 625 bp and 167 bp fragments of ALV-A, -B and -J, respectively, and showed no cross-reactivity with irrelevant pathogens, suggesting the excellent specificity of the assay. The constructed standard DNA templates were used to estimate the limit of detection. As shown by the results, the detection limit of the nanoPCR assay was nearly 10 copies/μL. To further evaluate the detection ability of the assay, 186 clinical samples were detected using the nanoPCR assay, among which, 14 samples were confirmed as ALV positive; the results were further confirmed by sequencing. In conclusion, a highly specific and sensitive nanoPCR assay was successfully developed, which could be a useful tool for clinical diagnosis as well as for the discrimination of ALV-A, -B and -J.
Topics: Animals; Avian Leukosis Virus; Sensitivity and Specificity; Nanoparticles; Temperature; Polymerase Chain Reaction; Avian Leukosis; Chickens
PubMed: 38275950
DOI: 10.3390/v16010015 -
Human Gene Therapy Methods Jun 2019In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice...
Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.
In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 10 leukocytes (including 1.1-2.3 × 10 NK cells) collected by small-scale lymphapheresis ( = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS basal medium containing NK MACS supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.
Topics: Alpharetrovirus; Cell Engineering; Cell Line; Cell Survival; Cytokines; Genetic Vectors; Humans; Killer Cells, Natural; Quality Control; Receptors, Chimeric Antigen; Transduction, Genetic
PubMed: 30997855
DOI: 10.1089/hgtb.2019.039 -
Frontiers in Immunology 2019In this study, a novel oral vaccine of recombinant () containing the gp85 protein was explored, and the effects of this vaccine on the prevention of subgroup J Avian...
In this study, a novel oral vaccine of recombinant () containing the gp85 protein was explored, and the effects of this vaccine on the prevention of subgroup J Avian Leukosis Virus (ALV-J) infection were assessed. In the current study, the gp85 protein of ALV-J was expressed on the surface of with the surface-display motif, , by constructing a shuttle vector . Surface localization of the fusion protein was verified by western blotting and flow cytometry. Subsequently, Specific Pathogen Free Hy-Line Brown layer chickens were orally vaccinated with the recombinant and presented with high levels of serum immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA) titers in bile and duodenal-mucosal fluid. After challenged with ALV-J of a 3 × 10 50% tissue culture infective dose (TCID50), serum samples of the chickens were collected and viremia was analyzed. Results showed that, compared to the and PBS control group, the recombinant group showed a significant rise in antibody levels after inoculation, and provide improved protection against ALV-J according to viremia detection. These results indicate that oral immunization with the recombinant provided an effective means for eliciting protective immune response against early ALV-J infection.
Topics: Administration, Oral; Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; Lactobacillus plantarum; Microorganisms, Genetically-Modified; Poultry Diseases; Viral Envelope Proteins; Viral Vaccines
PubMed: 31632395
DOI: 10.3389/fimmu.2019.02299 -
The Journal of Experimental Medicine Apr 2021In 1911, more than a century ago, Peyton Rous described a curious observation, later explained by a virus named for him that led to the discovery of oncogenes, the...
In 1911, more than a century ago, Peyton Rous described a curious observation, later explained by a virus named for him that led to the discovery of oncogenes, the modern era of cancer research, and the emergent field of precision medicine (1911. J. Exp. Med. https://doi.org/10.1084/jem.13.4.397).
Topics: Animals; Chickens; History, 20th Century; Humans; Molecular Targeted Therapy; Nobel Prize; Oncogenes; Precision Medicine; Rous sarcoma virus; Sarcoma, Avian
PubMed: 33710257
DOI: 10.1084/jem.20201754 -
BMC Veterinary Research Jun 2020Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection...
BACKGROUND
Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection on the intestinal microflora of chickens.
RESULTS
A 29-week commercial layer flock positive for avian leukosis virus-J (ALV-J), Marek's disease virus (MDV) and avian reticuloendotheliosis virus (REV) was selected, and fresh fecal samples were collected and examined for the composition of the gut microflora by Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The operational taxonomic units (OTUs) of the fecal microbiota differentiated the chickens infected with only ALV-J and those coinfected with ALV-J and MDV or REV from infection-negative chickens. The enrichment and diversity of cloacal microflora in chickens infected with ALV-J alone were slightly different from those in the infection-negative chickens. However, the diversity of cloacal microflora was significantly increased in chickens coinfected with both ALV-J and MDV or REV.
CONCLUSIONS
The intestinal microbiota was more strongly disturbed in chickens after coinfection with ALV-J and MDV or REV than after infection with ALV-J alone, and there may be underlying mechanisms by which the capacity for the stabilization of the intestinal flora was impaired due to viral infection and tumorigenesis.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Bacteria; Chickens; Coinfection; Feces; Female; Gastrointestinal Microbiome; Herpesvirus 2, Gallid; Marek Disease; Poultry Diseases; RNA, Ribosomal, 16S; Reticuloendotheliosis virus; Retroviridae Infections; Tumor Virus Infections
PubMed: 32600312
DOI: 10.1186/s12917-020-02430-3 -
Journal of Veterinary Science May 2020Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic...
BACKGROUND
Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components.
OBJECTIVES
The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV.
METHODS
Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction.
RESULTS
The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells.
CONCLUSIONS
Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.
Topics: Actin Cytoskeleton; Animals; Avian Leukosis; Avian Leukosis Virus; Avian Proteins; Coinfection; Gene Expression Regulation; Poultry Diseases; Reticuloendotheliosis virus; Retroviridae Infections; Tripartite Motif Proteins
PubMed: 32476322
DOI: 10.4142/jvs.2020.21.e49