First point of attack: Understanding the entry mechanism of SARS-CoV-2 into human cells

In a recent study published in the Journal of Extracellular Vesicles, researchers from Kanazawa University have visualized structural changes on the surface of SARS-CoV-2 that enable it to enter human cells.

The biology of SARS-CoV-2, the virus behind the COVID-19 pandemic, remains partially elusive. Understanding viral mechanisms is a key factor in developing effective treatment strategies against the outbreak. Now, Keesiang Lim and Richard Wong from Kanazawa University and colleagues have shown how the virus is equipped to enter human cells in real-time.

SARS-CoV-2 is enveloped by spike proteins, which form a crown-like layer on its surface. The immune system detects these spike proteins and prepares to neutralize the virus. Spike proteins also play a role in mediating entry of SARS-CoV-2 into cells. To-date, scientists have been able to take high-resolution albeit stationary images of spike proteins. Importantly, Richard Wong’s team and Kanazawa University used an advanced form of microscopy to capture dynamic changes in spike proteins when bound to cells.

Spike proteins comprise two major components—a globular head (that has a host recognition domain) attached to a stalk (that is capable of fusing with cells and facilitating entry). The researchers used high-speed atomic force microscopy (HS-AFM) to understand this structure deeper using just single molecules of spike proteins.

“We previously showed that the real-time observation of structural dynamic of influenza A hemagglutinin during viral entry,” says lead author of the study Dr. Keesiang Lim.

In the newly-published study, they found that the stalk showed a very flexible nature with an ability to extend or retract whereas the head could change conformation resulting in disappearance of the host recognition domain. Spike proteins typically latch onto cells that exhibit a molecule called ACE2 on their surface. Thus, the interactions of spike proteins with ACE2 were then visualized by HS–AFM. It was found that spike proteins docked onto ACE2 with the host recognition domain exposed. What’s more, their elastic nature enabled a far smoother interaction.

Small extracellular vesicles (sEV) are sacs released by cells which are composed of the similar chemical constituents as the cell membrane. The dynamics of spike proteins on sEVs was studied next. Since the spike protein stalk facilitates binding and fusion with membranes, interactions of only the stalk with sEVs were analyzed first. Indeed, a disruption of sEV membranes was seen indicating the stalk could fuse with cell membranes easily. However, when the dynamics of the entire spike protein were assessed, stable binding was only observed with sEVs released from ACE2-containing cells. ACE2 was thus a key factor in mediating viral entry.

HS-AFM proved to be a very useful tool in understanding the entry mechanics of SARS–CoV-2 in detail.

“Altogether, our study provides a platform that is suitable for real-time visualization of various entry inhibitors, neutralizing antibodies, and sEV-based decoy in blocking viral entry,” explains Dr. Richard Wong, senior author of the study. “Blocking the connections between spike proteins and ACE2 or inhibiting the membrane disruption caused by the spike protein stalk could be potential strategies preventing SARS–CoV-2 from hijacking the body.”

Real-time interaction between the SARS-CoV-2 Spike S2 subunit and sEVs

(a-e) A representative HS-AFM visualization of S2 subunit-sEV interaction (n: 9). (a) Multiple S2 subunits interacted with sEVs for different lengths of time. The durations of the interactions were recorded and categorized into three groups. Data are presented in bar graphs. (b) HS-AFM images show a S2 subunit (indicated by a white arrow) in contact with a sEV and subsequent insertion into the sEV (scale bar: 100 nm). Graphs beneath the HS-AFM images indicate the distance between the S2 subunit and the sEV during the interaction (red arrow: sEV, blue arrow: S2 subunit). (c) A kymograph shows a S2 subunit stably bound onto a sEV surface and eventually inserting into the sEV lipid layer. (d) Real-time height (h) and length (l) changes in the S2 subunit throughout the interaction with sEVs. The changes 6 s before contact (Free) and 6 s before insertion (Inserted) are illustrated in different line graphs. (e) Longer term observations of the S2 subunit–sEV interaction revealed that insertion of the S2 subunit disrupted the integrity of the exosomal layer, as indicated by a declining trend in sEV height. (f) Disruption of the exosomal shape in S2 subunit–sEV premixed samples. The S2 subunit was found to be attached to the sEV surface after incubation for 20 min and the sEV lost its spherical shape after 2 h of incubation with S2 subunit. A control sEV maintained a perfect spherical shape (scale bar: 50 nm)


ACE2: Angiotensin-converting enzyme 2, or ACE2, is a protein found on the membrane of cells located in the upper respiratory tract, intestines, kidneys, heart and other organs. The physiologic role of ACE2 is to metabolize hormones and stimulate their function.

ACE2 is also a docking point for several coronaviruses. The virus-ACE2 complex is engulfed into the cell providing an easy method of entry for the pathogen. What’s more, SARS-CoV-2 is known to bind more efficiently to ACE2 than SARS–CoV-1 which was responsible for the SARS epidemic. Understanding the dynamics of SARS–CoV-2 and ACE2 interactions are thus vital to developing strategies to prevent viral entry.

Small extracellular vesicles (sEV): The cells in our body release small vesicles that enable them to transport biomolecules, communicate with other cells and release signals when pathogens are detected. sEVs are a subset of such vesicles with a very small particle size. These vesicles are created when the membrane of a cell pinches off into smaller sac-like structures. The membranes of sEVs thus closely resemble those of living cells.

Since sEVs are released in various infections and cancer they also are being investigated as therapeutic targets. For example, ACE2 containing sEVs could be used as bait to trap SARS-CoV-2 and subsequently neutralize the virus.


Lim K, Nishide G, Yoshida T, Watanabe-Nakayama T, Kobayashi A, Hazawa M, Hanayama R, Ando T, Wong RW. (2021) Millisecond dynamic of SARS-CoV-2 spike and its interaction with ACE2 receptor and small extracellular vesicles. J Extracell Vesicles 10(14):e12170. [article]

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