D614/G614 variants of SARS-CoV-2 affect neuronal transmission; G614 better at membrane fusion

The COVID-19 pandemic is showing a strong comeback after an initial reduction in the number of cases, following energetic public health interventions in most parts of the world.

Overexpression of SARS2 spike protein alters the density and morphology of dendritic spines of cultured neurons. (

Overexpression of SARS2 spike protein alters the density and morphology of dendritic spines of cultured neurons. (A) Mouse cortical neurons were cotransfected with GFP and either vector control or HA-tagged D614 spike protein at DIV13. Five days later, immunostaining was performed to monitor GFP and HA signals. Counterstaining with DAPI was included to label the nuclei of neurons. Red arrows point to some very tiny spine heads. Scale bar: upper, 20 µm; lower, 5 µm.

During the research, it became clear that the virus had changed its ancestral genotype, developing several clades. Among these, the variant with the D614G mutation has rapidly risen to dominance wherever it was introduced. An interesting study published in the pre-print server bioRxiv* attempts to explain the neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection as well as the higher transmissibility of this variant.

Viral Fusion with Host Cell

The virus engages host cells via its receptor-binding domain (RBD), on its spike S1 subunit, which binds to the angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors. This triggers fusion events between the viral and host cell membranes, allowing the virus to enter the cytoplasm of the cell and begin replication.

The fusion occurs also between the cell membranes of adjacent cells, causing the formation of syncytia with multiple nuclei. The distribution of ACE2 over a wide range of cell types allows the virus to infect multiple organs, including the olfactory epithelium and the neurons of the central nervous system. Brain organoid cultures and animal studies have confirmed the presence of SARS-CoV-2 genomes and proteins in neurons.

Neurological Manifestations of SARS-CoV-2

Both in adults and infants with SARS-CoV-2 infection, some show neurological manifestations including olfactory and gustatory dysfunction, headache, dizziness, fluctuations of consciousness, seizures, inflammation of the CNS, and very uncommonly, ataxia or seizures. Neuropsychiatric syndromes like delirium or psychosis have also been reported.

Long-haul COVID-19 symptoms are being recognized, persisting in apparently recovered patients, indicating the virus may leave its mark on the CNS for a long period. Brain organoid studies hint at the underlying mechanism, namely, death of neurons and low neuronal protein markers. Protein-protein interactions between the virus and the host cell may alter the cellular states, affecting neuronal function.

Study Details

To understand the role of the D614G mutation in increased infectivity, the researchers explored the characteristics of the original D614 and the later G614 variants.

They found that both variants infect cultured neurons and are widely distributed within them. The presence of the spike protein was associated with higher dendritic spine density, narrowing of the spine heads, and longer spines overall. The latter two changes in the spine morphology indicate functional defects, which suggests synaptic transmission is impaired by the infection. This in turn causes neurological symptoms.

The SARS-CoV-2 spike RBD was used to elicit seven monoclonal antibodies in a mouse model. Two of these were used in the next phase of the experiment. The antibodies detected the viral spike protein specifically, in the MBP-RBD fusion protein, and also recognized the full-length spike protein tagged with a C-terminal HA molecule. They also detected a 120-kDa band, thought to be the post-cleavage S1 fragment of the SARS-CoV-2 spike.

Thus, these could be potent detection tools for discovering the expression, distribution within the subcellular components, and proteolysis of the viral spike protein within host cells.

Spike protein mutations may also affect the transmissibility of the virus. Therefore, the researchers also studied the different cell lines for proteolytic cleavage of the spike subunit. They found that the Neuro-2A cell lines showed higher G614 relative to D614.

Moreover, the different cell lines had varying amounts of S1 and S2 fragments, which suggests the trafficking and cleavage of the spike within the endoplasmic reticulum (ER) and Golgi apparatus are different within different cells.

Higher Infectivity

Among possible reasons for higher transmission, the researchers observed that the G614 shows greater proteolysis than the original variant, and thus generates more S2 subunits. Secondly, some of the glycosylated, full-length, or S1 fragment variants of the former mutation seemed to be phosphorylated after translation, which may confer these differing characteristics.

Since these produce higher levels of S2 subunits, this may explain the dominance of G614, since it is more efficient at fusion.

Higher Syncytial Formation

Indeed, when cells expressing human ACE2 and spike protein, respectively, are cultured together, the number of syncytia formed was greater when G614 spike was expressed relative to D614. Moreover, the number of syncytia with 4 or more nuclei was also larger in this group.

This suggests more effective cell fusion with G614, which allows more efficient viral entry and greater fusion of the infected cell with adjacent non-infected cells. This alteration could be the reason for the emergence of this strain as the dominant isolate worldwide.

They also found that with both spike variants, S1 and S2 spike protein fragments have different distributions. While the S1 is abundant at the ER, the S2 is found to cluster at the cell surface and the filopodia. The presence of S2 near filopodia may enhance the efficiency of syncytia formation, promoting the spread of the virus to nearby cells, and impair the function of the host cell.

Synaptic Impairment

The S2 fragments of both variants, interestingly, showed similar activity in reducing synaptic transmission and altering synapse shape by shortening the length of the dendrites of developing neurons. Thus, dendritic length and spines are affected, which probably impairs synaptic function.

What are the Implications?

Our study sheds light on the properties of SARS2 spike protein in host cells. Our findings also provide possible explanations for why the D614G mutation enhances infectivity and how SARS2 infection impairs neuronal function.”

Future electrophysiological studies are required to confirm this hypothesis. The research could also focus on the role played by spike proteins in the regulation or targeting of F-actin, which is a cytoskeletal protein that is crucial to neuronal morphology.

The authors put forth three possible mechanisms of neuronal infection by SARS-CoV-2. One is infection via low ACE2 titers, because of high viral concentrations. Secondly, the infection could be mediated by the NRP1 receptor, present at high levels on nerve cells. Or else, digested fragments of S2 on the viral surface and the surface of infected cells could trigger membrane fusion, allowing viral particles in the adjacent infected cells to enter.

In this scenario, infected cells could fuse with nearby neurons to introduce the virus too. These could work independently or in coordination with each other, and deserve further study.

*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.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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