Mechanistic insights on how the SARS-CoV-2 virus influences, modifies, and produces neuropathology in the nervous system

A recent research published in the journal Science Advances found that infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and viral fusogenic produce glial and neuronal fusion.

Infections in the neurological system can be caused by fungi, bacteria, viruses, and parasites. Viruses that can infect neurons include the Zika virus, reovirus, SARS-CoV-2, and herpes simplex virus.

Fever, loss of smell or taste, epileptic convulsions, disorientation, headaches, and, in extreme cases, meningitis, encephalitis, paralysis, and death can all result from viral brain infections.

Reoviruses and enveloped viruses employ fusogenic to fuse with host cellular membranes, penetrate cells, and hijack cellular machinery to produce viral components in non-neuronal tissues.

The newly formed fusogens redecorate the cell membrane, allowing it to merge with neighboring cells, resulting in multinucleated syncytia. It is uncertain if viral infection and fusogenic trigger neuronal fusion and syncytia development.

The research and its findings
The current study looked at whether SARS-CoV-2 infection and viral fusogenic produce neuronal cell fusion. First, plasmids expressing human angiotensin-converting enzyme 2 (hACE2) and green fluorescent protein (GFP) were transfected into a population of mouse brain cells.

A second group received plasmids encoding hACE2 and mCherry. These populations were co-plated and incubated for five days in vitro.

Infected cultures received 20, 2 x 103, or 2 x 106 plaque-forming units of ancestral SARS-CoV-2. Confocal microscopy was used to evaluate the cultures 72 hours after inoculation. Fused neurons were discovered, and antibody labeling verified the cells’ spike-positive status. The researchers also discovered new fusion phenotypes, like as glia-glia and glia-neuron fusions.

Only at higher SARS-CoV-2 titers was cell damage detected. SARS-CoV-2 was then transmitted into brain organoids generated from human embryonic stem cells (hESCs). Similarly, neuronal syncytia were seen in these infected organoids.

Furthermore, the scientists employed a SARS-CoV-2 spike and the baboon orthoreovirus p15 fusion protein to assess the impact of fusogenic alone.

A population of mouse neurons was transfected with plasmids expressing p15 and GFP. Another group was transfected with an empty vector and a mCherry plasmid. The populations were co-plated and cultured together. p15 expression was sufficient to produce neuronal fusion, which was not seen in the absence of p15.

The use of an inactive p15 entirely eliminated fusion. These procedures were then replicated with the SARS-CoV-2 spike protein. One neuronal group received spike- and GFP-expressing plasmids, whereas another received mCherry- and hACE2-expressing plasmids.

The populations were co-plated and cultured for 72 hours. The fusion of spike-expressing cells with hACE2-expressing cells was reported. Fusion required hACE2 as well as a spike. The use of inactive spike variants did not result in fusion. Both p15 and spike fusogens produced glia-glia and glia-neuron fusion phenotypes.

The researchers next looked into whether fusogenic might cause fusion in vivo. Vectors expressing GFP alone or GFP plus p15 were injected into the cortex and hippocampus of 11-week-old mice, and brains were removed seven and fourteen days later. Neuronal fusion has been observed in the cortex and hippocampus.

They next examined the SARS-CoV-2 spike or p15 in human-derived neurons. GFP and spike, inactive spike, or p15 were transfected into hESC-derived cortical neurons and neural progenitor cells. Three days later, p15 revealed clusters of linked GFP-positive neuronal cells, comparable to the syncytia seen in mouse neurons.

When the SARS-CoV-2 spike was produced, clusters formed, showing that fusion may occur via endogenous hACE2 receptors. There was no fusion with the inactive spike or without fusogens. The researchers then investigated if fusion was feasible at the neurite level, away from the cell bodies.

Infection with SARS-CoV-2 caused fusion between somas as well as between neurites remote from somas. Fusion-produced bridges with lengths that exceeded hundreds of micrometers. The researchers saw mitochondria and a fluorescent protein (cardinal) interact over the fusion bridges.

Over the course of seven days, neurons transfected with GFP and p15 were monitored for syncytia development. In the presence of p15, syncytia grew over time, with the gradual inclusion of additional cells. Finally, the researchers looked into how fusion affected neural activity.

To do this, differentiated neurons were fused with p15 and the fusion was seen using a fluorescent indicator sensitive to calcium ions. Neurons expressing mCherry and an empty vector were active spontaneously. The majority of fused neurons (90%) had synchronized neuronal activity, whereas the remainder displayed total cessation of neuronal activity.

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Cells with no activity were those that united securely at the soma level. All neurons united with glia had stopped working. Regardless of the synchronization of fused neurons, the frequency of neural activity remained constant. The researchers also discovered elevated intracellular calcium ion levels within the bridges.

The researchers discovered that virus-infected neurons or those expressing viral fusogenic might merge with neighboring neurons and glia, altering neuronal transmission and impairing neuronal function. SARS-CoV-2 neuronal fusion is dependent on hACE2 expression and potentially other auxiliary proteins.

Large molecules and organelles were also exchanged as a result of fusion. Furthermore, united neurons were functional, albeit with altered function and circuitry. The majority of currently available SARS-CoV-2 vaccines, including BNT162b2, ChAdOx1, mRNA-1273, and Ad26.COV2.S relies on spike expression in host cells to generate an immunological response.

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The full-length spike is encoded in these vaccines, with two mutations that stabilize the prefusion conformation and inhibit their fusogenicity. In the current study, the researchers employed the identical variant of the dormant spike that failed to cause fusion.

According to the findings, evaluating the fusogenic potential would be critical in developing future SARS-CoV-2 vaccines.

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