SARS-CoV-2 moves between permissive cells and non-permissive neuronal cells via tunneling nanotubes (TNT)

In a recent study published on the preprint server bioRxiv*, scientists investigate the neuroinvasive potential of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the role of tunneling nanotubes (TNTs) in spreading the infection to neighboring cells. The findings from this study reveal that neuronal cells are not permissive to SARS-CoV-2 through an exocytosis/endocytosis dependent pathway; however, they can be infected when co-cultured with permissive infected epithelial cells.

Using cryo-electron tomography, the scientists revealed that the SARS-CoV-2 induces TNT formation between cells and subsequently exploits this route to invade uninfected cells in co-culture.

Study: Tunneling nanotubes provide a novel route for SARS-CoV-2 spreading between permissive cells and to non-permissive neuronal cells. Image Credit: Alexander Limbach /


Differential neurological manifestations associated with the coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 infection, have been reported. Although acute neurological symptoms may eventually resolve, these symptoms have often been reported to persist during long-COVID. To date, it remains unclear how SARS-CoV-2 gains access to the central nervous system (CNS).

SARS-CoV-2 binds its spike protein to the host cell’s surface angiotensin-converting receptor 2 (ACE2) to gain entry. ACE2 receptors are found in organs such as the lungs, intestines, liver, kidneys, and heart. However, its expression is low in the human brain, with the exception of certain areas such as the thalamus and choroid plexus. Despite the overall lack of ACE2 receptors in the brain, SARS-CoV-2 has been found to propagate throughout the brain ain brain tissues of COVID-19 decedents.

In a previous study, scientists have shown a novel mechanism of cell-to-cell communication by spreading different amyloid aggregates between the CNS and from the peripheral lymphoid system cells to neurons via TNTs.

TNTs are dynamic connections between cells that consist of thin membranous conduits rich in actin that form contiguous cytoplasmic bridges between cells over long and short distances. These TNTs are capable of transporting cargo, including viruses, between cells.

Several recent studies have also reported the formation of TNTs or TNT-like structures that have been induced by retroviruses, herpesviruses, influenza A, and the human metapneumovirus to enable the efficient spread of infection to neighboring healthy cells. As a result of these previous observations, the scientists of the current study investigated the TNTs role in the neuroinvasive potential of SARS-CoV-2.

Importantly, the scientists noted that when the virus is transferred through the TNTs, viruses escape neutralizing antibody activity and immune surveillance. These viruses are also able to invade less permissive cells, such as those lacking the receptor for virus entry. Thus, the viruses favorably induce TNT formation to allow for the spread of virus tropism and pathogenicity.

Study findings

To understand how the SARS-CoV-2 infects neuronal cells, the researchers tested the permissivity of several different cell types to viral infection by the receptor-mediated pathway. The cell lines used in this study included human colon epithelial cell lines (Caco-2,), monkey kidney epithelial cell-line (Vero E6), and the human (SH-SY5Y) and murine (CAD) neuronal cells lines.

The scientists found that only the epithelial Vero E6 and Caco-2 cells, rather than the neuronal cells, were susceptible to infection by SARS-CoV-2. However, upon co-culturing, they found that the virus was transferred by a cell-to-cell contact-dependent mechanism and actively replicated in the neuronal cells. Thus, the scientists established that the SARS-CoV-2 can spread among cells through an exocytosis/endocytosis independent pathway.

Probing the direct cell-to-cell contact-dependent manner of the viral spread in neuronal cells, the scientists tested whether the virus triggered the formation of the TNTs or TNT-like structures in the infected cells and uses these structures to efficiently spread to uninfected cells. Using confocal microscopy, the scientists confirmed that TNTs contribute to the SARS-CoV-2 transmission.

To identify the nature and structure of the viral particles shared by TNTs and their mechanism of transfer, the scientists set up a correlative fluorescence and cryo-electron microscopy and tomography approach (CLEM, cryo-EM, and cryo-ET, respectively).

Strikingly, the images revealed viral compartments in TNTs. This included the presence of membranous structures of various sizes resembling double-membrane vesicles inside the tube, between permissive (Vero E6 infected cells) and non-permissive cells (SH-SY5Y mCherry cells).

Further, they also confirmed that these TNTs facilitate SARS-CoV-2 transmission between the permissive Vero E6 cells through a secretion-independent pathway. Between these permissive cells, the researchers found that the SARS-CoV-2 particles decorating the TNTs’ surface-displayed both an ellipsoidal- and spherical-enveloped morphology with an average diameter ranging from 50 to 100 nanometer (nm), typically of a coronavirus. Here, the cryo-EM images revealed SARS-Cov-2 on top of the TNTs, which was unlike the observation in the neuronal cells.

Significance of the study

The current study provides new information on the structure of the viral particles involved in intercellular spreading and provides important information about the mechanism of SARS-CoV-2 infection and transmission in neuronal cells.

Further, this study shows the remarkable role of the induced TNTs in the viral transmission in both permissive and non-permissive cells, possibly enhancing the efficiency of viral propagation through the body. The scientists state this report represents the first evidence that TNTs could be one possible route for the spreading of SARS-CoV-2.

*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. Ramya Dwivedi

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.


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