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A real-time molecular investigation of coronavirus entry into human cells

Susheela Srinivas

The COVID-19 pandemic has set off a wave of research activities across the world, aimed at finding clues that would allow us to design effective therapeutics and vaccines. In one such effort, a team of researchers from the Indian Institute of Technology, Kanpur, have initiated a study into the molecular dynamics of the process via which the novel coronavirus attaches to cells of the human respiratory system. 

SARS Co V 2 Illustration
An artist's rendering of SARS-CoV-2 entering human cells (Image: We Are Covert via Wikimedia Commons)

The novel coronavirus (SARS-CoV‑2), that causes the highly infectious disease COVID-19, has an outer layer of spiky protein molecules that helps it invade cells of the human respiratory system. A team of researchers led by Dibyendu Kumar Das, from the Indian Institute of Technology (IIT), Kanpur, is using specialised imaging techniques to peek into the molecular-level events that occur when the virus gains entry into the host cell.

By visualising the mechanism of viral entry into cells in real-time, the researchers hope to find clues that would allow us to devise strategies for battling the disease. 

Das, a Wellcome Trust/​DBT India Alliance Intermediate Fellow, has previously worked on the Ebola and Influenza viruses. For studying the novel coronavirus, Das and his team are employing a technique called Single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) to observe rapid changes in the viral spike proteins at the molecule level. 

Global research efforts have already revealed that the novel coronavirus is an enveloped virus. As the name implies, enveloped viruses have an outer covering that envelopes’ their genetic material. The envelope of the new coronavirus has special spike-like proteins called S‑glycoproteins that help the virus anchor to the host cell. 

S‑glycoprotein, in turn, is made up of three smaller protein subunits called S‑proteins, each with two domains — S1 and S2. The S1 domain latches on to human cells by binding to a protein called ACE2. The S2 domain then links the membranes of the virus and human cells, undergoing structural changes to help form a pore through the membrane. Next, the virus’s genetic material (RNA) passes through this pore into the host cell, allowing it to multiply inside the human cells. 

What remains unknown, however, is exactly how S2 fuses the two membranes. Das and his team plan to investigate this by imaging the S‑glycoprotein directly while the virus is making its entry into the host cell. The study will also allow them to understand how this protein interacts with cells of the respiratory system as well as immune cells.

The researchers chose the technique of sm-FRET to investigate this process based on Das’s use of this tool for his previous work on enveloped viruses. We found it to be a powerful probe to observe the dynamic changes in macromolecules – it shows how differently each spike molecule is behaving from the other,” explains Das.

Das explains the strategy they are planning to follow, which relies on the property of fluorescence and a phenomenon called resonance energy transfer’. The researchers will tag the S‑glycoprotein of the virus with a fluorescent dye, called the donor’ tag. At the same time, the researchers will also place an acceptor’ tag on ACE2, the protein on the human cell membrane that the virus docks to. When the donor’ and acceptor’ tags come close to one another, and the two are excited by a laser, the donor transfers some energy to the acceptor, changing the nature of light energy emitted by both tags. This energy transfer depends on the physical separation between the donor and acceptor tags. 

So when the S‑glycoprotein latches onto ACE2 and begins to change its molecular structure to allow the membranes to fuse, the events can be recorded in terms of changes in the spectral output (nature and intensity of fluorescence) of the donor and acceptor tags. A high resolution, highly sensitive camera called EMCCD will capture the minute sequential changes during the process. By measuring these changes, the researchers can gain insights into the dynamics of membrane fusion by the S‑glycoprotein.

Insights into the molecular mechanisms via which viruses invade host cells can be highly useful in designing therapeutic or preventive strategies against infectious diseases like COVID-19. The insights will facilitate designing structure dynamics- based vaccines, drugs and diagnostics for COVID19,” says Das.