Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.
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http://dx.doi.org/10.1369/0022155415593323 | DOI Listing |
J Virol
December 2024
National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, Wuhan, China.
is a relatively new viral family that was established nearly 5 years ago, but their viral morphologies (naked or encapsidated) remain controversial since only one member namely, filamentous virus 1 (CcFV1), was identified as being encapsidated in filamentous virions. Here, three novel double-stranded RNA (dsRNA) viruses belonging to the family were identified in three phytopathogenic fungal strains and tentatively named -sinensis polymycovirus 1 (PcsPmV1), and polymycovirus 1 and 2 (PhcPmV1 and 2), respectively. PcsPmV1 and PhcPmVs have five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, encoding five or seven putative open reading frames (ORFs), of which ORF1 encodes an RNA-dependent RNA polymerase, ORF5 encodes a prolein-alanine-serine-rich (P-A-S-rich) protein behaving as coat protein (CP); and dsRNAs 4 and 6 encode putative proteins with unknown functions and share no detectable identities with known viral sequences.
View Article and Find Full Text PDFEMBO J
December 2024
Institut Pasteur, Advanced Molecular Virology Unit, Department of Virology, Université Paris Cité, 75015, Paris, France.
Entry of viral capsids into the nucleus induces the formation of biomolecular condensates called HIV-1 membraneless organelles (HIV-1-MLOs). Several questions remain about their persistence, in vivo formation, composition, and function. Our study reveals that HIV-1-MLOs persisted for several weeks in infected cells, and their abundance correlated with viral infectivity.
View Article and Find Full Text PDFInt J Biol Macromol
January 2025
Department of Wood Anatomy and Utilization, Research Institute of Wood Industry, Chinese Academy of Forestry, Beijing 100091, China; Wood Specimen Resource Center (WOODPEDIA) of National Forestry and Grassland Administration, Beijing 100091, China. Electronic address:
Pharmaceuticals (Basel)
November 2024
Airway Disease, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG, UK.
Cilomilast, a phosphodiesterase-4 (PDE4) selective inhibitor, has anti-inflammatory effects in vitro and in vivo and reduces COPD exacerbations. We tested the hypothesis that cilomilast inhibits virus-induced airway epithelial intercellular adhesion molecule-1 (ICAM-1) expression and inflammatory cytokine/chemoattractants, IL-6, CXCL8, and CCL5 production in vitro. BEAS-2B bronchial epithelial cells were incubated with 0.
View Article and Find Full Text PDFAnal Chem
December 2024
Key Lab of Land Resources Evaluation and Monitoring in Southwest, Ministry of Education, College of Chemistry and Materials Science, Sichuan Normal University, Chengdu, Sichuan 610068, China.
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