A fast open-source Fiji-macro to quantify virus infection and transfection on single-cell level by fluorescence microscopy

Kerkhoff, Yannic; Wedepohl, Stefanie; nxiong, Nie Chua; Ahmadi, Vahid; Haag, Rainer; Stephan, Block

doi:10.1016/j.mex.2022.101834

Cited by 5 publications

(3 citation statements)

References 22 publications

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“…The inocula were removed, and cells were washed and incubated in complete media for an additional 7 h. Cells were then fixed using 10% formalin and processed for immunofluorescence using an antibody against the HCoV-OC43 N protein (Millipore #MAB9013; diluted 1:500) and a secondary Alexa Fluor 555 conjugated anti-mouse IgG antibody (NEB #4413S; diluted 1:1000). The percentage of infected cells was calculated using an open-source Fiji macro (83).…”

Section: Methodsmentioning

confidence: 99%

Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

Siwak,

LeBlanc,

Scott

et al. 2024

Preprint

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Cell entry of severe acute respiratory coronavirus-2 (SARS-CoV-2) and other CoVs can occur via two distinct routes. Following receptor binding by the spike glycoprotein, membrane fusion can be triggered by spike cleavage either at the cell surface in a transmembrane serine protease 2 (TMPRSS2)-dependent manner or within endosomes in a cathepsin-dependent manner. Cellular sialoglycans have been proposed to aid in CoV attachment and entry, although their functional contributions to each entry pathway are unknown. In this study, we used genetic and enzymatic approaches to deplete sialic acid from cell surfaces and compared the requirement for sialoglycans during endosomal and cell-surface CoV entry, primarily using lentiviral particles pseudotyped with the spike proteins of different sarbecoviruses. We show that entry of SARS-CoV-1, WIV1-CoV and WIV16-CoV, like the SARS-CoV-2 omicron variant, depends on endosomal cathepsins and requires cellular sialoglycans for entry. Ancestral SARS-CoV-2 and the delta variant can use either pathway for entry, but only require sialic acid for endosomal entry in cells lacking TMPRSS2. Binding of SARS-CoV-2 spike protein to cells did not require sialic acid, nor was sialic acid required for SARS-CoV-2 entry in TMRPSS2-expressing cells. These findings suggest that cellular sialoglycans are not strictly required for SARS-CoV-2 attachment, receptor binding or fusion, but rather promote endocytic entry of SARS-CoV-2 and related sarbecoviruses. In contrast, the requirement for sialic acid during entry of MERS-CoV pseudoparticles and authentic HCoV-OC43 was not affected by TMPRSS2 expression, consistent with a described role for sialic acid in merbecovirus and embecovirus cell attachment. Overall, these findings clarify the role of sialoglycans in SARS-CoV-2 entry and suggest that cellular sialoglycans mediate endosomal, but not cell-surface, SARS-CoV-2 entry. Thus, it may be important to consider both cell entry pathways when developing sarbecovirus entry inhibitors targeting virus-sialoglycan interactions.Author summaryThe COVID-19 pandemic, caused by SARS-CoV-2, has resulted in over 676 million infections and 6.8 million deaths so far, demonstrating the threat posed by emerging CoVs. In humans, SARS-CoV-2 and related coronaviruses cause respiratory tract infections, such as the common cold, as well as more severe disease in some individuals. To prepare for future outbreaks, conserved steps in the CoV replication could be considered for antiviral prophylactic or therapeutic approaches. One such process is CoV cell entry, which occurs via two main routes: At the cell surface or within endosomes. Cellular receptors, proteases and complex sugars, known as glycans, mediate CoV entry steps. In this study, we compared the role of a specific glycan subset, sialoglycans, in endosomal and cell surface CoV entry. We show that sialoglycans are required for entry of various CoVs that are mainly dependent on the endosomal route, but in the case of SARS-CoV-2, sialoglycans were not required when the cell-surface entry route was available. Our findings contribute to understanding the mechanisms of CoV entry, which could inform development of pan-CoV antivirals that target CoV entry steps.

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Section: Methodsmentioning

confidence: 99%

Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

Siwak,

LeBlanc,

Scott

et al. 2024

Preprint

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“…The time-dependent infection for sulfated hydrogels (Figure 7b) was automatically analyzed based on the DAPI and GFP signal (Figure 7b). [64] Time dependent live cell microscopy images for MH-S-4_1. 25 showed extremely slow infection rate (Figure 7d).…”

Section: Synthetic Hydrogels As Protective Barrier Against Hsv-1 Infe...mentioning

confidence: 99%

Mucus-Inspired Self-Healing Hydrogels: A Protective Barrier for Cells against Viral Infection

Bej,

Stevens,

Nie

et al. 2024

Preprint

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Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad spectrum of viruses. Here we developed a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ~10 % repeating units of activated disulfide as cross-linking sites. Cryo-EM analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as HSV-1 and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched PEG-thiol as cross-linkers. Single particle tracking microrheology, EPR and Cryo-SEM were used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. a transwell assay was used to investigate the hydrogel’s protective properties against viral infection against HSV-1. Live-cell microscopy confirmed that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, our novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as a models for disulfide-rich airway mucus research, and as biomaterials.

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“…The time-dependent infection for sulfated hydrogels was automatically analyzed based on the GFP signal (Figure 7b). [67] The analysis for MH-S-4_1. 25 showed extremely slow infection rate (Figure 7b and Figure S29, Supporting Information).…”

Section: Synthetic Hydrogels As Protective Barrier Against Hsv-1 Infe...mentioning

confidence: 99%

Mucus‐Inspired Self‐Healing Hydrogels: A Protective Barrier for Cells against Viral Infection

Bej,

Stevens,

Nie

et al. 2024

Advanced Materials

Self Cite

View full text Add to dashboard Cite

Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad spectrum of viruses. Here we developed a polyglycerol sulfate‐based dendronized mucin‐inspired copolymer (MICP‐1) with ∼10% repeating units of activated disulfide as cross‐linking sites. Cryo‐EM analysis of MICP‐1 reveals an elongated single‐chain fiber morphology. MICP‐1 shows potential inhibitory activity against many viruses such as HSV‐1 and SARS‐CoV‐2 (including variants such as Delta and Omicron). MICP‐1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched PEG‐thiol as cross‐linkers. Single particle tracking microrheology, EPR and Cryo‐SEM were used to characterize the network structures. The synthesized hydrogels exhibit self‐healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay was used to investigate the hydrogel's protective properties against viral infection against HSV‐1. Live‐cell microscopy confirmed that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, our novel mucin‐inspired copolymer generates mucus‐mimetic hydrogels on a multi‐gram scale. These hydrogels can be used as a models for disulfide‐rich airway mucus research, and as biomaterials.This article is protected by copyright. All rights reserved

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A fast open-source Fiji-macro to quantify virus infection and transfection on single-cell level by fluorescence microscopy

Cited by 5 publications

References 22 publications

Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

Mucus-Inspired Self-Healing Hydrogels: A Protective Barrier for Cells against Viral Infection

Mucus‐Inspired Self‐Healing Hydrogels: A Protective Barrier for Cells against Viral Infection

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