In a recent study posted to the bioRxiv* preprint server, researchers performed the photocatalytic mapping of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein-host cell membrane interactions.
Understanding viral entrance and pathogenicity can be improved by identifying protein habitats at the virus-host cell interface. The virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2, uses the angiotensin-converting enzyme-2 (ACE2) protein as a primary receptor. However, the role of other cellular proteins in the entrance process is uncertain.
About the study
In the present study, the team developed a viral-host protein microenvironment mapping technology (ViraMap) using iridium photocatalysts (IrPC) conjugated to SARS-CoV-2 spike protein for visible-light-driven proximity labeling on host cells.
The team generated spike protein photocatalyst conjugates for targeted labeling on ACE2-expressing cells to analyze the interactions in the SARS-CoV-2 spike-host cell surface microenvironment using ViraMap. The SARS-CoV-2 spike protein trimer and its associated variants, including the SARS-CoV-1 spike and SARS-CoV-2 D614G spike, were chosen for conjugation with IrPC.
Further, the researchers performed targeted labeling on the cells by exposing them to spike-IrPC conjugates in the presence of a biotin-diazirine probe. This was followed by blue light irradiation and subsequent monitoring with flow cytometry and confocal imaging analysis. To selectively label host cell surface contacts and avoid cellular internalization of spike-IrPC conjugates, HEK293T+ACE2 cells were treated with spike-IrPC at 4 °C.
The team assessed cell surface binding in the presence of free ACE2 protein to establish that the spike-IrPC compound preserved its affinity for ACE2 (ACE2-Fc). For cell surface proximity labeling on HEK293T+ACE2 cells, three spike-IrPC variants such as SARS-CoV-1, SARS-CoV-2, and SARS-CoV-2 D614G spike proteins, were employed.
Furthermore, the team used a primary-secondary antibody combination comprising an anti-ACE2 primary antibody and an antibody-photocatalyst conjugate for targeted labeling. After extraction from the cell membrane fraction and streptavidin enrichment, the biotinylated host proteins from these targeted labeling studies were quantitatively characterized using a tandem mass tag (TMT)-based liquid chromatography–mass spectrometry (LC-MS)/MS quantification.
Results
When compared to a non-binding antibody immunoglobulin G (IgG) control conjugated with IrPC, flow cytometry analysis revealed a clear change in the biotinylation signal for spike-IrPC variant conjugates-treated cells. Compared to the mouse-IgG-IrPC control, confocal imaging of spike-IrPC induced labeling revealed biotin localisation to the host cell surface environment. Furthermore, because the recombinant spike construct had a poly-His-tag, cellular biotinylation might be achieved through targeted labeling with an anti-His antibody IrPC conjugate.
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IrPC-conjugated SARS-CoV-1, SARS-CoV-2, SARS-CoV-2 D614G spike proteins, as well as anti-ACE2 primary/secondary antibody systems resulted in statistically significant enrichment of distinct groups of cellular proteins compared to the IrPC isotype conjugate as a negative control. Using the SAINTexpress and mass spectrometry interaction statistics (MiST) scoring algorithms, the team identified 96 high-confidence enriched proteins across all spike-IrPC labeling studies.
Almost 23 of the 96 high-confidence enriched proteins were shared by all three spike variants, whereas the remaining two, 25, and 46 enriched proteins were unique to the SARS-CoV-1, SARS-CoV-2, and SARS-CoV-2 D614G spike-IrPC spikes, respectively. The gene ontology enrichment analysis of the 23 enriched proteins found a significant relationship with viral entrance, followed by immune cell differentiation and co-stimulation activities. Furthermore, 70% of the enriched proteins had a high abundance in most human tissues, including the lung, kidney, gastrointestinal tract, fat, cardiac muscle, soft tissues, reproductive tissues, and brain.
Out of 23, a total of 10 high-confidence enriched proteins could be divided into two groups based on whether or not they showed co-enrichment with the anti-ACE2 primary/secondary antibody system. The first class of cell surface receptors included ACE2, neuropilin 1 (NRP1), prostaglandin F2 receptor negative (PTGFRN), and neurogenic locus notch homolog protein 2 (NOTCH-2). Evidence of PTGFRN protein and messenger ribonucleic acid (mRNA) expression in a wide range of tissue cells, particularly cardiomyocytes and lung fibroblasts, implied that these cell types are highly susceptible to SARS-CoV-2 via various entry points. Galectins were the second set of host proteins found in all three spike-IrPC labeling assays.
The results of the targeted labeling experiments point to putative cell surface co-receptors involved in viral entry and antiviral immunity. As a result, functional experiments were performed to assess which of the identified host proteins are involved in SARS-CoV-2 entrance into cells. For functional validation, the study focused on the ten overlapping proteins shared by all three spike-IrPC variants. The clustered regularly interspaced short palindromic repeats /Cas9-mediated knockdown (KD) only reduced ACE2 abundance by 30%. Although there was a 40% reduction in protein expression relative to controls, there was no significant change in pseudoparticle entrance following NRP1 KD.
Overall, the study findings showed that the ViraMap technology successfully facilitated the assessment of known as well as unknown virus-host protein interactions that occur on the outer cell membrane of the host cell using cell surface recognition targeting modalities.
*Important notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Some Ontario doctors have started offering a free shot that can protect babies from respiratory syncytial virus while Quebec will begin its immunization program next month.
The new shot called Nirsevimab gives babies antibodies that provide passive immunity to RSV, a major cause of serious lower respiratory tract infections for infants and seniors, which can cause bronchiolitis or pneumonia.
Ontario’s ministry of health says the shot is already available at some doctor’s offices in Ontario with the province’s remaining supply set to arrive by the end of the month.
Quebec will begin administering the shots on Nov. 4 to babies born in hospitals and delivery centers.
Parents in Quebec with babies under six months or those who are older but more vulnerable to infection can also book immunization appointments online.
The injection will be available in Nunavut and Yukon this fall and winter, though administration start dates have not yet been announced.
This report by The Canadian Press was first published Oct. 21, 2024.
-With files from Nicole Ireland
Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.
ISLAMABAD (AP) — Polio cases are rising ahead of a new vaccination campaign in Pakistan, where violence targeting health workers and the police protecting them has hampered years of efforts toward making the country polio-free.
Since January, health officials have confirmed 39 new polio cases in Pakistan, compared to only six last year, said Anwarul Haq of the National Emergency Operation Center for Polio Eradication.
The new nationwide drive starts Oct. 28 with the aim to vaccinate at least 32 million children. “The whole purpose of these campaigns is to achieve the target of making Pakistan a polio-free state,” he said.
Pakistan regularly launches campaigns against polio despite attacks on the workers and police assigned to the inoculation drives. Militants falsely claim the vaccination campaigns are a Western conspiracy to sterilize children.
Most of the new polio cases were reported in the southwestern Balochistan and southern Sindh province, following by Khyber Pakhtunkhwa province and eastern Punjab province.
The locations are worrying authorities since previous cases were from the restive northwest bordering Afghanistan, where the Taliban government in September suddenly stopped a door-to-door vaccination campaign.
Afghanistan and Pakistan are the two countries in which the spread of the potentially fatal, paralyzing disease has never been stopped. Authorities in Pakistan have said that the Taliban’s decision will have major repercussions beyond the Afghan border, as people from both sides frequently travel to each other’s country.
The World Health Organization has confirmed 18 polio cases in Afghanistan this year, all but two in the south of the country. That’s up from six cases in 2023. Afghanistan used a house-to-house vaccination strategy this June for the first time in five years, a tactic that helped to reach the majority of children targeted, according to WHO.
Health officials in Pakistan say they want the both sides to conduct anti-polio drives simultaneously.
WASHINGTON (AP) — Millions of people with private health insurance would be able to pick up over-the-counter methods like condoms, the “morning after” pill and birth control pills for free under a new rule the White House proposed on Monday.
Right now, health insurers must cover the cost of prescribed contraception, including prescription birth control or even condoms that doctors have issued a prescription for. But the new rule would expand that coverage, allowing millions of people on private health insurance to pick up free condoms, birth control pills, or “morning after” pills from local storefronts without a prescription.
The proposal comes days before Election Day, as Vice President Kamala Harris affixes her presidential campaign to a promise of expanding women’s health care access in the wake of the U.S. Supreme Court’s decision to undo nationwide abortion rights two years ago. Harris has sought to craft a distinct contrast from her Republican challenger, Donald Trump, who appointed some of the judges who issued that ruling.
“The proposed rule we announce today would expand access to birth control at no additional cost for millions of consumers,” Health and Human Services Secretary Xavier Becerra said in a statement. “Bottom line: women should have control over their personal health care decisions. And issuers and providers have an obligation to comply with the law.”
The emergency contraceptives that people on private insurance would be able to access without costs include levonorgestrel, a pill that needs to be taken immediately after sex to prevent pregnancy and is more commonly known by the brand name “Plan B.”
Without a doctor’s prescription, women may pay as much as $50 for a pack of the pills. And women who delay buying the medication in order to get a doctor’s prescription could jeopardize the pill’s effectiveness, since it is most likely to prevent a pregnancy within 72 hours after sex.
If implemented, the new rule would also require insurers to fully bear the cost of the once-a-day Opill, a new over-the-counter birth control pill that the U.S. Food and Drug Administration approved last year. A one-month supply of the pills costs $20.
Federal mandates for private health insurance to cover contraceptive care were first introduced with the Affordable Care Act, which required plans to pick up the cost of FDA-approved birth control that had been prescribed by a doctor as a preventative service.
The proposed rule would not impact those on Medicaid, the insurance program for the poorest Americans. States are largely left to design their own rules around Medicaid coverage for contraception, and few cover over-the-counter methods like Plan B or condoms.
