Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

Rensen, Elena; Pietropaoli, Stefano; Mueller, Florian; Weber, Christian; Souquère, Sylvie; Sommer, Sina; Isnard, Pierre; Rabant, Marion; Gibier, Jean‐Baptiste; Terzi, Fabiola; Simon‐Lorière, Etienne; Rameix‐Welti, Marie‐Anne; Pierron, Gérard; Barba-Spaeth, Giovanna; Zimmer, Christophe

doi:10.26508/lsa.202101124

Cited by 21 publications

(23 citation statements)

References 46 publications

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“…We hypothesized that SARS-CoV-2 could use TNTs to spread from permissive cells to less-permissive cells that lack the membrane receptor for virus entry, thus allowing the spreading of viral pathogenicity and escaping from immune surveillance. To test this hypothesis, we used the Vero E6 cell line as an epithelial model because it has been widely used for SARS-CoV-2 isolation, propagation, and antiviral testing (36)(37)(38). As a neuronal model of nonpermissive cells, we used the SH-SY5Y cell line; these are human cells widely used as a neuronal model, and their TNTs have also been thoroughly characterized (39) and are identifiable with high reliability (39,40).…”

Section: Introductionmentioning

confidence: 99%

Tunneling nanotubes provide a route for SARS-CoV-2 spreading

et al. 2022

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Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain. We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells. SARS-CoV-2 induces the formation of tunneling nanotubes (TNTs) and exploits this route to spread to uninfected cells. In cellulo correlative fluorescence and cryo–electron tomography reveal that SARS-CoV-2 is associated with TNTs between permissive cells. Furthermore, multiple vesicular structures such as double-membrane vesicles, sites of viral replication, are observed inside TNTs between permissive and nonpermissive cells. Our data highlight a previously unknown mechanism of SARS-CoV-2 spreading, likely used as a route to invade nonpermissive cells and potentiate infection in permissive cells.

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Section: Introductionmentioning

confidence: 99%

Tunneling nanotubes provide a route for SARS-CoV-2 spreading

et al. 2022

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“…Therefore, Huh-7 cells were incubated with AKS466, infected with 50 µL SARS-CoV-2 for 24 h, fixed, and permeabilized. Viral RNA genomes and mRNAs are detected with complementary labelled fluorescence oligonucleotides using confocal fluorescence imaging, according to Rensen et al [ 25 ]. SARS-CoV-2-infected cells show two specific types of (+) RNA fluorescence signals.…”

Section: Resultsmentioning

confidence: 99%

“…FISH of SARS-CoV-2-positive RNA-strand was performed, as described recently [ 18 ]. Briefly, 93 primary oligos were ordered in 96 well format (Merck-Sigma Aldrich (Taufkirchen, Germany), at a concentration of 100 µM and mixed equimolar; for oligonucleotide sequences see Supplementary Information .…”

Section: Methodsmentioning

confidence: 99%

The Acid Ceramidase Is a SARS-CoV-2 Host Factor

Geiger

Kersting

Schlegel

et al. 2022

Cells

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SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.

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“…Studying SARS-CoV-2 with fluorescence microscopy has been recently discussed elsewhere [ 89 ]. However, FISH and Flow-FISH have also been employed to study SARS-CoV-2 [ 88 , 90 ] (see Table 6 ). If available, probe sequences and limits of detection can be found in Supplementary Table S6 .…”

Section: Flow-fish Applications In Microorganismsmentioning

confidence: 99%

“…If available, probe sequences and limits of detection can be found in Supplementary Table S6 . In a recent unreviewed preprint, FISH was used to detect SARS-CoV-2 with a probe set that targeted the conserved regions of the SARS-CoV-2 genome [ 90 ]. Authors were able to detect SARS-CoV-2 mRNA in cell lines, postmortem patient tissue samples, and nasal swabs typically used for SARS-CoV-2 diagnostic purposes.…”

Section: Flow-fish Applications In Microorganismsmentioning

confidence: 99%

Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses

Heeren¹

2021

BioTech

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Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2.

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Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

Cited by 21 publications

References 46 publications

Tunneling nanotubes provide a route for SARS-CoV-2 spreading

Tunneling nanotubes provide a route for SARS-CoV-2 spreading

The Acid Ceramidase Is a SARS-CoV-2 Host Factor

Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses

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