Furin cleavage sites in the spike proteins of bat and rodent coronaviruses: Implications for virus evolution and zoonotic transfer from rodent species

Abstract

Bats and rodents comprise two of the world's largest orders of mammals and the order Chiroptera (bats) has been implicated as a major reservoir of coronaviruses in nature and a source of zoonotic transfer to humans. However, the order Rodentia (rodents) also harbors coronaviruses, with two human coronaviruses (HCoV-OC43 and HCoV-HKU1) considered to have rodent origins. The coronavirus spike protein mediates viral entry and is a major determinant of viral tropism; importantly, the spike protein is activated by host cell proteases at two distinct sites, designated as S1/S2 and S2'. SARS-CoV-2, which is considered to be of bat origin, contains a cleavage site for the protease furin at S1/S2, absent from the rest of the currently known betacoronavirus lineage 2b coronaviruses (Sarbecoviruses). This cleavage site is thought to be critical to its replication and pathogenesis, with a notable link to virus transmission. Here, we examine the spike protein across coronaviruses identified in both bat and rodent species and address the role of furin as an activating protease. Utilizing two publicly available furin prediction algorithms (ProP and PiTou) and based on spike sequences reported in GenBank, we show that the S1/S2 furin cleavage site is typically not present in bat virus spike proteins but is common in rodent-associated sequences, and suggest this may have implications for zoonotic transfer. We provide a phylogenetic history of the Embecoviruses (betacoronavirus lineage 2a), including context for the use of furin as an activating protease for the viral spike protein. From a One Health perspective, continued rodent surveillance should be an important consideration in uncovering novel circulating coronaviruses.

Keywords: Bats; Coronavirus; Furin; Rodents; Spike protein.

Fig. 1

Examples of predicted furin cleavage sites identified in coronaviruses associated with chiropteran species. Over 150 spike sequences from bat associated CoVs were screened for furin cleavage sites, including those in both the Alpha- and Betacoronavirus genera, using the programs PiTou and ProP. A. Unique S1/S2 furin cleavage sites predicted in bat associated coronaviruses. B. Unique S2’ furin cleavage sites predicted across bat associated coronaviruses. Associated NCBI accession numbers are as follows: Bat Hp-betacoronavirus/Zhejiang2013 (YP_009072440), (Putative) Zaria bat coronavirus (ADY17911), Bat coronavirus HKU5–1 (ABN10875), BtPa-BetaCoV/GD2013 (AIA62343), Pipistrellus abramus bat coronavirus HKU5-related (QHA24687), Bat coronavirus HKU5-2 (ABN10884), Pipistrellus bat coronavirus HKU5 (AGP04938), Coronavirus Neoromicia/PML-PHE1/RSA/2011 (AGY29650), BtCoV/A434/2005 (ABG11962), Bat coronavirus PREDICT/PDF-2180 (YP_009361857), BtRt-BetaCoV/GX2018 (QJX58383), Rousettus bat coronavirus HKU 9 (AVP25406), Bat coronavirus 1B (ACA52157), 229E-related bat coronavirus (ALK28781), BtMf AlphaCoV/GD2012-b (AIA62241), Bat coronavirus MfulBtCoV/3709 (AMB43191), Bat coronavirus MfulBtCoV/3736-1 (AMB43194), Bat coronavirus MfulBtCoV/3759-1 (AMB43195), Bat coronavirus MsBtCoV/4001-1 (AMB43196), Bat coronavirus MsBtCoV/4068 (AMB43198), Rhinolophus bat coronavirus HKU32 (QCX35178), Rousettus bat coronavirus GCCDC1 (QKF94914), Hipposideros bat coronavirus HKU10 (AFU92131), Bat coronavirus HKU9-3 (ABN10927), Bat coronavirus HKU9-1 (ABN10911), Alphacoronavirus Bat-CoV/P.kuhlii/Italy/3398-19/2015 (YP_009755890), NL63-related bat coronavirus (YP_009824967), Chaerephon bat coronavirus/Kenya/KY41/2006 (ADX59458), Miniopterus bat coronavirus/Kenya/KY33/2006 (ADX59488), SARS-like coronavirus BatCoV/BB9904/BGR/2008 (ALJ94036), and Rousettus bat coronavirus/Kenya/KY06/2006 (ADX59474).

Fig. 2

S1/S2 furin cleavage sites are commonly found in rodent associated coronaviruses. A. Examples of predicted S1/S2 furin cleavage sites in rodent associated coronaviruses. Longquan RI rat coronavirus Longquan-189 (AID16649) shared the same S1/S2 furin cleavage site as Longquan Rl rat coronavirus Longquan-370 (AID16655). Murine hepatitis virus strain JHM (YP_209233) shares the same S1/S2 furin cleavage sites as several other MHV strains (ACN89763, ACN89705, ACN89696, ACN89722), as well as Murine coronavirus MHV-3 (ACN89743) in which sites occur at residues 770 and 769, versus 769 and 768. Longquan Rl rat coronavirus Longquan-708 (AID16661) shares the same S1/S2 cleavage site as Murine coronavirus MHV-1 (ACN89742), in which the predicted furin cleavage site occurs at residue 758. Betacoronavirus HKU24 Lijiang-41 (QOE77297) shares the same S1/S2 furin cleavage site as Betacoronavirus HKU24 Lijiang-53 (QOE77307) and Betacoronavirus HKU24 Ruili-874 (QOE77327), in addition to several other accession numbers (AYR18625, AYR18679, AYR18634, AYR18607, AYR18652, AYR18670, and AYR18643). Coronavirus AcCoV-JC34 (YP_009380521) shares the same S1/S2 furin cleavage site as Lucheng Rn rat coronavirus Lijiang-170 (QOE77268), which were only predicted by PiTou. RtRt-CoV/Tb2018 (QIM73854) shared the same S1/S2 predicted cleavage sites as RtRt-CoV/Tk2011 (QIM73813). Rodent coronavirus RtMm-CoV/GD2015 (ATP66756) shared the same S1/S2 predicted cleavage site as Rodent coronavirus RtRn-CoV/YN2013 (ATP66727). RtRe-CoV/Tl2009 (QIM73841) shared the same S1/S2 furin cleavage site as RtRt-CoV/Tn2018 (QIM73848). B. S2’ furin cleavage sites predicted in rodent association coronaviruses, both of which were noted as unclassified, but our analysis supported as belonging to the Embecovirus subgenera. *Our analysis supports classification as an Embecovirus.

Fig. 3

Phylogenetic tree of spike protein sequences from the Embecovirus clade. A) The tree was constructed using RaxML [81], 1000 bootstrap replicates, from a spike protein alignment, that removed any ambiguously aligned positions using Gblocks [60]. Bootstrap support percentages for nodes receiving less than 75% are not shown. B) Sequences are colored based on the predicted furin cleavage score from PiTou.