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.
The Canadian government says it will donate up to 200,000 vaccine doses to fight the mpox outbreak in Congo and other African countries.
It says the donated doses of Imvamune will come from Canada’s existing supply and will not affect the country’s preparedness for mpox cases in this country.
Minister of Health Mark Holland says the donation “will help to protect those in the most affected regions of Africa and will help prevent further spread of the virus.”
Dr. Madhukar Pai, Canada research chair in epidemiology and global health, says although the donation is welcome, it is a very small portion of the estimated 10 million vaccine doses needed to control the outbreak.
Vaccine donations from wealthier countries have only recently started arriving in Africa, almost a month after the World Health Organization declared the mpox outbreak a public health emergency of international concern.
A few days after the declaration in August, Global Affairs Canada announced a contribution of $1 million for mpox surveillance, diagnostic tools, research and community awareness in Africa.
On Thursday, the Africa Centres for Disease Control and Prevention said mpox is still on the rise and that testing rates are “insufficient” across the continent.
Jason Kindrachuk, Canada research chair in emerging viruses at the University of Manitoba, said donating vaccines, in addition to supporting surveillance and diagnostic tests, is “massively important.”
But Kindrachuk, who has worked on the ground in Congo during the epidemic, also said that the international response to the mpox outbreak is “better late than never (but) better never late.”
“It would have been fantastic for us globally to not be in this position by having provided doses a much, much longer time prior than when we are,” he said, noting that the outbreak of clade I mpox in Congo started in early 2023.
Clade II mpox, endemic in regions of West Africa, came to the world’s attention even earlier — in 2022 — as that strain of virus spread to other countries, including Canada.
Two doses are recommended for mpox vaccination, so the donation may only benefit 100,000 people, Pai said.
Pai questioned whether Canada is contributing enough, as the federal government hasn’t said what percentage of its mpox vaccine stockpile it is donating.
“Small donations are simply not going to help end this crisis. We need to show greater solidarity and support,” he said in an email.
“That is the biggest lesson from the COVID-19 pandemic — our collective safety is tied with that of other nations.”
This report by The Canadian Press was first published Sept. 13, 2024.
Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.
HALIFAX – The Nova Scotia government says it could be months before it reveals how many people are on the wait-list for a family doctor.
The head of the province’s health authority told reporters Wednesday that the government won’t release updated data until the 160,000 people who were on the wait-list in June are contacted to verify whether they still need primary care.
Karen Oldfield said Nova Scotia Health is working on validating the primary care wait-list data before posting new numbers, and that work may take a matter of months. The most recent public wait-list figures are from June 1, when 160,234 people, or about 16 per cent of the population, were on it.
“It’s going to take time to make 160,000 calls,” Oldfield said. “We are not talking weeks, we are talking months.”
The interim CEO and president of Nova Scotia Health said people on the list are being asked where they live, whether they still need a family doctor, and to give an update on their health.
A spokesperson with the province’s Health Department says the government and its health authority are “working hard” to turn the wait-list registry into a useful tool, adding that the data will be shared once it is validated.
Nova Scotia’s NDP are calling on Premier Tim Houston to immediately release statistics on how many people are looking for a family doctor. On Tuesday, the NDP introduced a bill that would require the health minister to make the number public every month.
“It is unacceptable for the list to be more than three months out of date,” NDP Leader Claudia Chender said Tuesday.
Chender said releasing this data regularly is vital so Nova Scotians can track the government’s progress on its main 2021 campaign promise: fixing health care.
The number of people in need of a family doctor has more than doubled between the 2021 summer election campaign and June 2024. Since September 2021 about 300 doctors have been added to the provincial health system, the Health Department said.
“We’ll know if Tim Houston is keeping his 2021 election promise to fix health care when Nova Scotians are attached to primary care,” Chender said.
This report by The Canadian Press was first published Sept. 11, 2024.
ST. JOHN’S, N.L. – Newfoundland and Labrador‘s chief medical officer is monitoring the rise of whooping cough infections across the province as cases of the highly contagious disease continue to grow across Canada.
Dr. Janice Fitzgerald says that so far this year, the province has recorded 230 confirmed cases of the vaccine-preventable respiratory tract infection, also known as pertussis.
Late last month, Quebec reported more than 11,000 cases during the same time period, while Ontario counted 470 cases, well above the five-year average of 98. In Quebec, the majority of patients are between the ages of 10 and 14.
Meanwhile, New Brunswick has declared a whooping cough outbreak across the province. A total of 141 cases were reported by last month, exceeding the five-year average of 34.
The disease can lead to severe complications among vulnerable populations including infants, who are at the highest risk of suffering from complications like pneumonia and seizures. Symptoms may start with a runny nose, mild fever and cough, then progress to severe coughing accompanied by a distinctive “whooping” sound during inhalation.
“The public, especially pregnant people and those in close contact with infants, are encouraged to be aware of symptoms related to pertussis and to ensure vaccinations are up to date,” Newfoundland and Labrador’s Health Department said in a statement.
Whooping cough can be treated with antibiotics, but vaccination is the most effective way to control the spread of the disease. As a result, the province has expanded immunization efforts this school year. While booster doses are already offered in Grade 9, the vaccine is now being offered to Grade 8 students as well.
Public health officials say whooping cough is a cyclical disease that increases every two to five or six years.
Meanwhile, New Brunswick’s acting chief medical officer of health expects the current case count to get worse before tapering off.
A rise in whooping cough cases has also been reported in the United States and elsewhere. The Pan American Health Organization issued an alert in July encouraging countries to ramp up their surveillance and vaccination coverage.
This report by The Canadian Press was first published Sept. 10, 2024.
