Natural selection differences detected in key protein domains between non-pathogenic and pathogenic Feline Coronavirus phenotypes

Abstract

Feline Coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed Feline Enteric Coronavirus [FECV]), with around 12% developing into deadly Feline Infectious Peritonitis (FIP; Feline Infectious Peritonitis Virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV and FECV specific signals of positive selection. We analyzed full length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site, and the other within the fusion domain of Spike. We also found 15 sites subject to positive selection associated with FIPV within Spike, 11 of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were 14 sites (12 novel) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 furin cleavage site and adjacent C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype, and included 24 positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that is unlikely to be one singular "switch" mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

Figure 1.

FIPV-1 Spike (Spike-1) domain map and tertiary structure highlighting sites subject to natural selection. Sites are mapped to the protein domain map and PDB structure 6JX7 accession number FJ938054 (Yang et al., 2020). A) S1 and S2 subunits of Spike further separated into functional protein subdomains. Dashed vertical black lines delimit numbered RFPs, and are colored based on association of phenotype with inferred selection. The two sites identified by Contrast-FEL (Kosakovsky Pond et al., 2021) to be evolving differently between FIPV and FECV are depicted in cyan. Codon sites subject to adaptive evolution associated with the FIPV phenotype are depicted in red. FECV associated codon sites subject to adaptive evolution are represented in purple. Text labels for each domain: 0-domain; A domain; B domain, receptor binding motif (RBM); C domain; D domain; S1/S2 furin cleavage site (FCS); upstream helix (UH); S2’ cleavage site; fusion domain (FD) with fusion peptide (FP); heptad repeat region 1 (HR1); central helix (CH); connector domain (CD); heptad repeat region 2 (HR2); transmembrane domain (TM); cytoplasmic tail (CT). Amino acid indices are reported for each domain. B) Sites mapped to PDB 6JX7 (trimer) to visualize selected sites in 3D space. C) The monomer representation. NTD is highlighted in yellow, CTD in gold, FP in green, HR1 in white, and the rest of the S2 subunit in light pink.

Figure 2.

Spike-1 S1/S2 furin cleavage motif with the amino acid composition at critical sites involved in cleavage function (P6 to P1’ (Licitra et al., 2014)) for the FIPV and FECV sequences used in this study. The P6 and P5 sites were subject to directional selection in FECV sequences (highlighted in purple), and the P4 site was identified by the Contrast-FEL method (Kosakovsky Pond et al., 2021) (highlighted in cyan) to be evolving differently between the two phenotypes. Furin cleavage occurs between the P1 and P1’ site (Licitra et al., 2014), depicted with the red scissors.

Figure 3.

Convergent evolution detected at site 1058 within FIPV-1 Spike protein sequences by PCOC (Rey et al., 2018). Branches tested are highlighted in red. A Leucine (L) has arisen from a Methionine (M) in (15/18) FIPV sequences. Each leaf (tip) is annotated with the amino acid, accession number, and clinically diagnosed phenotype. The “FL” (FJ938057) represents an ambiguous base. CCoV-1 strain 23-03 Spike (KP849472) was used to root the tree.