We know that the novel coronavirus SARS-CoV-2 uses the Spike protein present on its outer envelope to enter our body cells. Studies have shown that the Spike protein consists of two subunits, S1 and S2. First, S1 recognises receptors ACE2 (angiotensin-converting enzyme 2) and NRP1 (neuropilin-1) on our cell membrane and latches with them (like a key in a lock).

After latching, S2, a bundle of six helical proteins, comes into play. The proteins in S2 rearrange their structure, stretch and elongate to reach the target – our body cell’s membrane. Once they reach the boundary, a segment of S2 called the fusion peptide (another protein part) is released, which acts as an anchor for S2, the virus and host cell membranes fuse.   Once this fusion happens, a pathway is created, through which the viral genetic material, RNA (ribonucleic acid), enters the cell, hijacks the cell machinery to replicate and causes infection.

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On the other hand, the SARS-CoV-2 is mutating to evolve into dominant strains. Scientists have observed that maximum mutations are happening in the Spike protein, helping it improve its ability to stick and fuse with host cells better. Such enhanced fusion ability resulted in D614G emerging as a dominant strain in 2020 with high transmission rates. In addition, the virus evolved further to the Alpha (UK), Beta (South Africa), and Delta (India) strains, each causing a new wave of pandemic, challenging the efficacy of the vaccines.

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The knowledge gaps

Although studies have given insights into the Spike protein’s structure and details of how it adheres to the host cell wall, we still do not know many things about the fusion process. For example, are just the receptors (ACE2 and NRP1) on our cells enough to facilitate the fusion of S2 with the cell wall? Is the virus taking advantage of any other cell process or cell substructures and utilising them to promote the fusion of the membranes? Where exactly is the virus finding an entry? How is the virus using chemicals in our cells to its benefit? And how is the virus modifying its sensitivity to these cell chemicals to help it emerge into dominant variants that fuse better with cells?

These are some knowledge gaps in our understanding of the evolving viral strains. From a scientific study viewpoint, re-creating the Spike-cell fusion sequence processes in the laboratory would immensely help scientific studies. The insights would help design better drugs and vaccines.

Researchers from the Department of Biological Sciences and Bioengineering, the Indian Institute of Technology Kanpur (IIT Kanpur), bridge this knowledge gap by setting up a ‘live demonstration’ of the Spike-cell fusion process in the laboratory. Their study helps us understand and visualise how the S2 sub-unit is interacting with the host cells and undergoing changes for fusion. Furthermore, their experiments reveal which cell components and chemicals are aiding the virus to emerge into highly infectious dominant strains.

Rigging up a live demo

Cells are complex machines made up of several small parts called organelles present inside and on the cell surface. Each organelle has specific duties to perform, and collectively, the cell functions. For example, receptors are gatekeepers and help bind to drugs or hormones; mitochondria are the cell’s energy bank; ribosomes manufacture proteins; and, so on. Cells also contain many charged particles or chemical ions that act as messengers to carry signals between organelles, trigger enzyme reactions and carry information to other cells.

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The IIT team investigated how the five strains of the virus – the original Wuhan variety, D614G, Alpha, Beta, and Delta – interacted with cell components. They chose to experiment with synthetic cells, called liposomes, to mimic real cells. Liposomes are vesicles with fat layers on which they could coat the required receptor proteins, giving them the freedom to add each component sequentially and as required for observations. They observed the fusion process response by using advanced spectroscopic techniques like fluorescence de-quenching, fluorescence anisotropy and FRET measurements.

The team coated the liposomes with ACE2 and NRP1 receptors and observed how the viral strains interacted with them. They noticed that adding only these two receptors was insufficient for the S2 Spike sub-unit to fuse with the host cell membrane. They then modified the surrounding medium between the virus and the vesicle by altering the pH of the solution. pH is a quantitative measure of how chemically acidic, alkaline (basic) or neutral a solution is. They noticed a low pH of around 4.6 (an acidic medium) improved the Spike activity.

Then the researchers introduced metallic ions of iron, magnesium, zinc, and calcium into the surrounding medium and determined if they facilitated the fusion.

They found the Spike protein was only active when there were calcium ions, and, as the ion levels increased, the Spike could fuse better with the host membrane. “Calcium ions triggered the release of the fusion peptide segment of S2, which helps restructure the proteins and find the target membrane. As a result, S2 rapidly changed its structure and fused with the host membrane,” says Dr Dibyendu Das, lead author of the study. In addition, there was a peak response to a particular level of ion concentration (500µM – micro Molar, a unit which gives the measure in number of moles of a substance in a unit volume).

However, surprisingly, the ability of S2 to fuse with the cell decreased sharply when calcium ion levels increased beyond that.

Dr Das explains, “This is because S2 proteins require some time to locate the target membrane and then release a fusion peptide. When calcium ions increased beyond the optimum level, they triggered a fast reaction, releasing the fusion molecule sooner.” Hence S2 is unable to reach the target, and as a result, the anchoring peptide falls off, S2 collapses back, and the fusion is inhibited.

Advantage virus

The researchers thus concluded that the Spike protein has mutated to favour an acidic environment and a high sensitivity to a particular concentration of calcium ions in the cells. The combined effect enhances the Spike protein’s ability to fuse with cells, making the virus highly infectious. In addition, they found that the Delta strain exhibited the maximum fusion ability of 90 per cent under these conditions.

Asked how these observations relate to the real-cell environment, Dr Das says: “Our body cells have organelles (component) called lysosomes present on the cell membrane. They clean up cell waste. Lysosomes have an acidic medium and the exact calcium ion level (500µM) for fusion. Therefore, they offer a favourable point of entry for the virus.”

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The researchers conducted another experiment in actual cells. They used a special molecule that targeted and disrupted the calcium ion concentration in lysosomes. They noticed that this immediately inhibited Spike’s fusion and, hence, the viral entry to cells.

Dr Das says the results offer the scope for an effective mode for drug or vaccine design. “If we can somehow target the lysosomes in body cells and find a way to manipulate the calcium ion channels in lysosomes, we can inhibit the virus’s entry into cells.”

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