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ORIGINAL RESEARCH article

Front. Virol.

Sec. Fundamental Virology

Volume 5 - 2025 | doi: 10.3389/fviro.2025.1612630

The spike S2 substitution, P804L, drives the adaptation of BANAL-20-236 to a broad range of Rhinolophus bat ACE2

Provisionally accepted
Luo  ChenLuo Chen1Alfredo  A Hinay, Jr.Alfredo A Hinay, Jr.1Shigeru  FujitaShigeru Fujita1Jumpei  ItoJumpei Ito1Hiroyuki  AsakuraHiroyuki Asakura2Mami  NagashimaMami Nagashima2Kenji  SadamasuKenji Sadamasu2Kazuhisa  YoshimuraKazuhisa Yoshimura2Kei  SatoKei Sato1,3*
  • 1Division of Systems Virology, Institute of Medical Science, The University of Tokyo, Tokyo, Kyoto, Japan
  • 2Tokyo Metropolitan Institute of Public Health, Tokyo, Japan
  • 3The University of Tokyo, Bunkyo, Tōkyō, Japan

The final, formatted version of the article will be published soon.

Understanding how bat coronaviruses circulate in wildlife, including bats in nature, can help assess the risks of cross-species transmission of potentially pathogenic viruses to humans. However, the evolutionary molecular mechanisms that enable sarbecoviruses to utilize diverse Rhinolophus bat ACE2 receptors remain poorly understood. In this study, we investigate the adaptive potential of BANAL-20-236—a bat sarbecovirus sharing 95.2% amino acid identity with SARS-CoV-2—in overcoming ACE2 compatibility barriers across various Rhinolophus bat species. Comparative replication kinetics revealed that BANAL-20-236 exhibits reduced fitness compared to SARS-CoV-2 in the cells expressing ACE2 from R. ferrumequinum, R. sinicus, and R. shameli. Serial passaging in these cells was selected for the mutants bearing substitutions in the S2 subunit, P804L and S876Y, which enhanced infectivity in R. sinicus and R. shameli ACE2. Both substitutions conferred increased infectivity in the cells expressing a variety of Rhinolophus ACE2. We show that the P804L substitution, located near the S2’ cleavage site, increased viral infectivity in a transmembrane serine protease 2 (TMPRSS2)-independent entry. Conversely, the increased infectivity by the S876Y substitution, which is closed to heptad repeat 1, is dependent on TMPRSS2. Our in vitro cell culture experiments suggest that the S2-driven evolution occurring in nature could facilitate the adaptation of bat coronaviruses to the diverse usage of bat ACE2.

Keywords: Coronavirus, Sarbecovirus, Rhinolophus bat, ACE2, spillover

Received: 16 Apr 2025; Accepted: 31 Jul 2025.

Copyright: © 2025 Chen, Hinay, Jr., Fujita, Ito, Asakura, Nagashima, Sadamasu, Yoshimura and Sato. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Kei Sato, Division of Systems Virology, Institute of Medical Science, The University of Tokyo, Tokyo, 6068507, Kyoto, Japan

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