Ability of bat severe acute respiratory syndromes-like coronavirus (WIV1) to utilize raccoon dog angiotensin-converting enzyme as receptor for cellular entry

Abstract

Objective To investigate the ability of bat severe acute respiratory syndromes (SARS)-like coronavirus (WIV1 strain) to utilize angiotensin-converting enzyme Ⅱ (ACE2) from raccoon dog as a receptor for cellular entry, and to explore the potential cross-species transmissibility of WIV1. Methods Plasmids encoding ACE2 molecules from different animals including human (Homo sapiens), civet cat (Paguma larvata), Chinese horseshoe bat (Rhinolophus sinicus) and raccoon dog (Nyctereutes procyonoides) were constructed, and then transfected into 293T cells. The expressions of ACE2 proteins in 293T were detected by Western blot with anti-C9 antibody. The pseudoviral infection systems of SARS coronavirus (Tor2 strain) and bat SARS-like coronavirus (WIV1 strain) were established to detect receptor activity of different animals’ ACE2 for WIV1 entry. The plasmid expressing transmembrane serine protease 2 (TMPRSS2) was constructed, and then transfected into T Rex 293 cells. The effect of TMPRSS2 on WIV1pp infectivity was examined by luciferase assay. Results ACE2 from raccoon dog, Chinese horseshoe bat, civet cat and human expressed well in 293T cells and supported cellular entry mediated by WIV1 S protein. Compared with pcDNA3.1 vector, ACE2 molecules from raccoon dog, Chinese horseshoe bat, civet cat and human increased the WIV1pp infection by thousands of times, all with significant differences in luciferase activity (t = 27.744, P < 0.001; t = 18.740, P < 0.001; t = 32.297, P < 0.001; t = 15.902, P < 0.001 ). Compared with the group without TMPRSS2, the expression of TMPRSS2 in target cells increased the infection mediated by WIV1-S protein by more than 10 folds, with significant difference in luciferase activity (t = 29.460, P < 0.001). Conclusions The ACE2 molecules from raccoon dog served as a functional receptor for cellular entry mediated by WIV1 spike protein, which was activated by the TMPRSS2 protease. Our findings herein suggest that there may exist a risk of multiple cross-species transmission of WIV1 among human, bat and raccoon dog. Key words: Bat severe acute respiratory syndromes-like coronavirus; WIV1; Raccoon dog; Angiotensin-converting enzyme Ⅱ; Transmembrane protease serine 2