N-terminomics of SARS-CoV-2

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COVID-19 Imaging

A better understanding of the interactions between SARS-CoV-2 and the host cells it infects could lead to new approaches to treat or prevent infections. Proteolysis is essential to the SARS-CoV-2 life cycle; two viral polyproteins must be cleaved to generate components of the viral replication/transcription complex. The polyproteins are snipped apart by two viral proteases, the papain-like protease (PLP) and main protease (Mpro).

PLP and Mpro also cleave cellular proteins, which may help the virus by modifying the activities of these targets. Protease inhibitors have demonstrated antiviral activity in cell culture and are a subject of anti-coronavirus drug development. To this end, understanding the complete set of proteolytic changes that occur upon viral infection would be important.

Meyer et al carried out an unbiased study of proteolysis of viral and cellular proteins during viral infection in cell culture. Proteins were extracted from infected and uninfected cells, differentially labeled, and analyzed by mass spectrometry in an N-terminomics approach that identified neo-N-termini. In doing so, several cellular targets of viral proteases were identified as well as new viral protein cleavage sites, which hold potential for future vaccine or antibody development.”

Figure 4g from Meyer et al. (2021) Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential. Licensed under CC BY 4.0. The Azure c600 was used to capture images of a chemiluminescent Western blot, demonstrating the cleavage of cellular proteins PAICS and GOLGA3 in transfected cells expressing SARS-CoV-2 Mpro.

To confirm their findings, both in vitro and cell-based assays were used to observe the process of SARS-CoV-2 proteases cleaving cellular proteins. Specifically, SARS-CoV-2 Mpro was expressed in cells with the subsequent cleavage of two target proteins being confirmed via chemiluminescent Western blotting imaged on an Azure c600.

The authors found that the cleavage of cellular proteins was important for viral infectivity. To test for this, RNAi was used to deplete the levels of 14 potential viral protease substrates. Surprisingly, reducing each of the 14 proteins resulted in reduced viral titers, suggesting that viral proteases target and cleave cellular proteins to increase pro-viral activities. Consistent with this, two inhibitors of cellular targets were found to potently inhibit infection of cells by SARS-CoV-2, indicating the activity of these cellular proteins is important to viral infectivity.

This study demonstrates the enormous potential of characterizing proteolysis in the context of viral infection to aid in the development of targeted anti-viral strategies.

In addition to chemiluminescence imaging, the Azure 600 imager provides multicolor fluorescence, white light, and two-channel near-infrared imaging of blots, gels, and more.

Intranasal Administration of Neutralizing IgA Increases SARS-CoV-2 Infection in a Hamster Model

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COVID-19 Western Blotting

Infectivity of SARS-CoV-2 is associated with high viral loads in the upper respiratory tract. Alpha and gamma immunoglobulins (IgA and IgG) both neutralize viral infections, with IgA significant in mucosal immunity and IgG predominantly affecting blood. Most studies focus on IgG in SARS-CoV-2 infection, but the role of IgA is less understood.

In a preprint shared on ResearchSquare, Zhou et al investigated whether intranasal IgA could protect against SARS-CoV-2 infection in Syrian hamsters. The study is currently under review so the current version is not peer-reviewed or published in a journal. The authors report that neutralizing IgA antibodies, applied intranasally before SARS-CoV-2 challenge, reduced the amount of virus in the lungs but increased viral infectivity in the nasal turbinate of the hamsters.

INTRANASAL ADMINISTRATION OF NEUTRALIZING IGA INCREASES SARS-COV-2 INFECTION IN A HAMSTER MODEL
Figure 5. B8-dIgA1 and B8-dIgA2 enhance SARS-CoV-2 infection via CD209. C. The CD209 or CD299 overexpressed-HEK 293T cells were pre-treated for 6 hours with 10 ng/ml of B8-dIgA1 or B8-dIgA2 or control dIgA1 or control dIgA2 or PBS, respectively, prior to SARS-CoV-2 infection (MOI: 0.05). Two days after infection, SARS-CoV-2 NP expression (green) was quantified by the mean fluorescence intensity (MFI) after anti-NP IF staining. Statistics were generated using student-t tests. *p<0.05; **p<0.01; ***p<0.001.

Methods used

In the course of this work, the authors used the Azure Sapphire Biomolecular Imager in viral neutralization assays and assays of enhanced infection via CD209. For each assay type, cells were grown in 96-well plates and infected with SARS-CoV-2. The media was removed, cells permeabilized and SARS-CoV-2 was detected with an rabbit anti-virus primary antibody followed by a goat anti-rabbit secondary antibody labeled with Alexa Fluor 488. The resulting fluorescent signal was detected on the Sapphire (Figure 5C).

The authors isolated and characterized 18 RBD-specific monoclonal antibodies from four COVID-19 patients. Five of these showed neutralizing activity against SARS-CoV-2. The one with the tightest binding to RBD was tested in the hamster model to see if intraperitoneal administration of the antibody before or soon after viral challenge would affect infection. It did decrease viral loads in the lungs but did not prevent infection in the nasal turbinate.

The group then engineered monomeric and dimeric IgA1 and IgA2 versions of the antibody and tested these in hamsters, either by intraperitoneal injection or nasal administration. The monomeric IgA1 but not IgA2 protected lungs, while neither prevented nasal infection. When dimeric versions of the IgA1 and IgA2 were tested, they were found to decrease infection in the lungs but increase infection and enhance damage in the nasal turbinate.

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Findings

The authors found that the IgA-mediated enhancement did not depend on the ACE2 receptor. Instead, the targets were dendritic cells expressing CD209, a lectin known to be a receptor for secretory IgA.

How SARS-CoV-2 enters host cells

To enter host cells, the receptor binding domain (RBD) of the SARS-CoV-2 spike protein binds to cellular ACE2 receptors. The spike protein is cleaved and the viral and cellular membranes fuse, allowing the virus into the cell. The spike protein is the target of current SARS-CoV-2 vaccines and is the major antigen target of antibodies in infected individuals. Additionally, RBD-specific IgA have been found in COVID-19 patients.

DISCOVER: Azure Sapphire Biomolecular Imager

In addition to fluorescence imaging, the Sapphire Biomolecular Imager provides densitometry, phosphor, multichannel fluorescence, chemiluminescence and white light imaging of blots, gels, tissues, and more.