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

KANAZAWA, Japan, Dec. 8, 2021 /PRNewswire/ -- 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."

Background

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.

Reference

Keesiang Lim, Goro Nishide, Takeshi Yoshida, Takahiro Watanabe- Nakayama, Akiko Kobayashi, Masaharu Hazawa, Rikinari Hanayama, Toshio Ando, Richard W. Wong. Millisecond dynamic of SARS-CoV-2 spike and its interaction with ACE2 receptor and small extracellular vesicles. Journal of Extracellular Vesicles, 2021.

DOI:  10.1002/jev2.12170
https://doi.org/10.1002/jev2.12170

Related figures

https://nanolsi.kanazawa-u.ac.jp/wp-content/uploads/2021/12/fig-1.jpg
Figure 1. The molecular dynamic of the spike protein (Spike/S) of the coronavirus SARS-CoV-2 has been successfully observed directly for the first time in the world using HS-AFM.

https://nanolsi.kanazawa-u.ac.jp/wp-content/uploads/2021/12/fig-2.jpg
Figure 2: Overview: Direct visualisation of the interaction of coronavirus spike proteins with angiotensin converting enzyme 2 (ACE2) binding in real time using HS-AFM.

Contact 
Hiroe Yoneda
Vice Director of Public Affairs
WPI Nano Life Science Institute (WPI-NanoLSI)
Kanazawa University
Kakuma-machi, Kanazawa 920-1192, Japan
Email: nanolsi-office@adm.kanazawa-u.ac.jp
Tel: +81 (76) 234-4550

About Nano Life Science Institute (WPI-NanoLSI)

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Nano Life Science Institute (NanoLSI), Kanazawa University is a research center established in 2017 as part of the World Premier International Research Center Initiative of the Ministry of Education, Culture, Sports, Science and Technology. The objective of this initiative is to form world-tier research centers. NanoLSI combines the foremost knowledge of bio-scanning probe microscopy to establish 'nano-endoscopic techniques' to directly image, analyze, and manipulate biomolecules for insights into mechanisms governing life phenomena such as diseases.

About Kanazawa University

http://www.kanazawa-u.ac.jp/e/

As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.

The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas.