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. 2021 Jun;30(11):2607-2625.
doi: 10.1111/mec.15910. Epub 2021 May 3.

The virus-host interface: Molecular interactions of Alphacoronavirus-1 variants from wild and domestic hosts with mammalian aminopeptidase N

Ximena A Olarte-Castillo  1   2 , Joana F Dos Remédios  3 , Felix Heeger  1   4 , Heribert Hofer  1   5   6 , Stephan Karl  1 , Alex D Greenwood  1   5 , Marion L East  1   2
Affiliations

The virus-host interface: Molecular interactions of Alphacoronavirus-1 variants from wild and domestic hosts with mammalian aminopeptidase N

Ximena A Olarte-Castillo et al. Mol Ecol. 2021 Jun.

Abstract

The Alphacoronavirus-1 species include viruses that infect numerous mammalian species. To better understand the wide host range of these viruses, better knowledge on the molecular determinants of virus-host cell entry mechanisms in wildlife hosts is essential. We investigated Alphacoronavirus-1 infection in carnivores using long-term data on Serengeti spotted hyenas (Crocuta crocuta) and molecular analyses guided by the tertiary structure of the viral spike (S) attachment protein's interface with the host receptor aminopeptidase N (APN). We sequenced the complete 3'-end region of the genome of nine variants from wild African carnivores, plus the APN gene of 15 wild carnivore species. Our results revealed two outbreaks of Alphacoronavirus-1 infection in spotted hyenas associated with genetically distinct canine coronavirus type II (CCoVII) variants. Within the receptor binding domain (RBD) of the S gene the residues that directly bind to the APN receptor were conserved in all variants studied, even those infecting phylogenetically diverse host taxa. We identified a variable region within RBD located next to a region that directly interacts with the APN receptor. Two residues within this variable region were under positive selection in hyena variants, indicating that both sites were associated with adaptation of CCoVII to spotted hyena APN. Analysis of APN sequences revealed that most residues that interact with the S protein are conserved in wild carnivores, whereas some adjacent residues are highly variable. Of the variable residues, four that are critical for virus-host binding were under positive selection and may modulate the efficiency of virus attachment to carnivore APN.

Keywords: aminopeptidase N; carnivores; coronavirus; human APN; spike protein; virus-host interaction.

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Figures

FIGURE 1
FIGURE 1
Schematic representation of the complete 3′‐end of the Alphacoronavirus‐1 genome of variants from spotted hyena (SH, in pink) and silver‐backed jackal (SBJ, in blue) from 2001 to 2012. Variant SBJ12 2007 (on top) had all nine genes and therefore was set as a reference variant. Each box represents a gene and the name of each gene is indicated above the reference variant. The number inside each box represents the length of different genes in the reference variant. In other variants, gene length is only presented for genes that differed in size to genes in the reference variant. Hatched boxes represent deletions, the number of nucleotides (nt) deleted is indicated below each hatched box. The total size of each 3′‐end genome fragment, from the start of the S gene to the end (excluding the poly(A) tail), is indicated on the right of each schematic representation. The two SH variants from 2007 (SH33‐2007 and SH110‐2007) had the same genome arrangement. Three SH variants from 2011 and 2012 and one SBJ variant from 2011 (SH89‐2011, SH143‐2011, SH157‐2012 and SBJ3‐2011) had the same genome arrangement. Only the complete S, N and 7a genes were obtained from variant SH1‐2008
FIGURE 2
FIGURE 2
Similarity plot of the 3′‐end of the genome of six “Serengeti” variants. Four variants from spotted hyena (SH) and two from silver‐backed jackal (SBJ) against one spotted hyena variant from 2011 (SH143‐2011). SH variants include one from 2001 in green, one from 2004 in blue, and two from 2007 in yellow. The SBJ variants include one from 2007 in red and one from 2011 in brown. The locations of gene regions (S1‐NTD, S1‐CTD, S2, 3a, 3c, E, M, N) are indicated above the plot. The location of the receptor binding region (RBD) is indicated above the X axis. The plot was constructed with genes that did not have major deletions (see Figure 1)
FIGURE 3
FIGURE 3
The phylogenetic relationships of “Serengeti” variants and other Alphacoronavirus‐1 variants based on the S1‐CTD domain of the S gene (1161 nt) and the distribution across lineages of two residues (524, 525) under positive selection. The “Serengeti” variants from spotted hyenas (SH) are in pink and those from silver‐backed jackals (SBJ) are in blue. For each variant, the coronavirus genotype, variant name, host, country of origin, year of collection and Genbank accession number are quoted. Numbers at the branches indicate bootstrap percentage values from 1000 replicates. Branch colour indicate evidence of significant episodic positive selection (EBF >50) for site 524 (in orange) and selective sweeps (R → L, EBF >100) for site 525 (in green)
FIGURE 4
FIGURE 4
The phylogenetic relationships of the APN gene from 34 mammalian species and the distribution across lineages of five residues (735–738, 784) critical for the interaction between APN and the Alphacoronavirus‐1 virus receptor binding region, four of which are under positive selection. Maximum likelihood tree under the HKY85 + G + I model of a segment of 1941 nucleotides (nt) of the APN nt sequence. The fifteen carnivore APN sequences obtained by this study are in bold letters. The family for each species is indicated to the right of the tree, followed by the suborder to which each carnivore species belongs. Numbers at the branches indicate bootstrap percentage values from 1000 replicates. Accession numbers of each sequence are specified after each species name. Residue 735 is under positive selection. Residues 736, 738 and 784 are under episodic diversifying selection. Branches in green, red and light blue indicate where in the tree are residues 736, 738 and 784 under episodic diversifying selection, respectively. On the right of the tree is the alignment of these five residues (735–738, 784). At the top of the alignment is the sequence of these residues in porcine APN (pAPN). Residues from other mammalian species APN which are identical to those in the domestic pig APN are highlighted in yellow, residues that differed are labelled with the respective amino acid. The plus signs indicate the four residues found under positive selection
FIGURE 5
FIGURE 5
Alignment of the region in the porcine APN (pAPN) protein known to bind to Alphacoronavirus‐1 of APN sequences from 18 carnivore species and the domestic pig. In bold are the 15 sequences obtained in this study, including four that are incomplete (African civet, serval, tiger, leopard) and those of the spotted hyena (in pink) and the silver‐backed jackal (in blue). Above the alignment is the representation of the tertiary structure (alpha helices) of pAPN, the black circles below indicate the 23 residues in pAPN that directly contact PRCV. Residues highlighted in yellow are identical to the ones observed in pAPN and residues that differ from those in pAPN are in a different colour to increase clarity. Plus signs indicate the eight residues under positive selection. Highlighted in cyan are four residues under positive selection that are not among the 23 residues in pAPN that interact with the porcine Alphacoronavirus‐1 PRCV
FIGURE 6
FIGURE 6
Model of the tertiary structure of porcine APN (pAPN) coupled with the RBD of the porcine Alphacoronavirus‐1 PRCV. The alpha helices and beta barrels are numbered in each structure. (a) For the pAPN structure, the region known to interact with PRCV is highlighted in green and the rest of the structure is in light grey. The specific residues known to interact with PRCV are coloured according to the number of different residues observed between APN of carnivore species included and the domestic pig (19 sequences in total), i.e., 0 indicates that the residue at a site was identical in all species and four indicates four different residues occurred in the species examined. Most residues were identical in all sequences (0, in magenta), including residues 736 to 738 (indicated in the structure) which are essential for the interaction with PRCV. Residue 735 was detected to be under positive selection and together with residue 739 was the most variable residue (4, light blue). (b) For the RBD structure, the two protruding regions that directly interact with pAPN (β1–β2, β3–β4) are marked in yellow. Within these two regions the four most important residues that interact with pAPN (527, 528, 530, 571) are shown and are identical in all variants studied. Residues detected under positive selection are coloured in red and most are outside the regions that directly interact with the receptor. Residues 524 (orange) and 525 (green) which were under some kind of selection in most Serengeti hyena variants are also shown
FIGURE 7
FIGURE 7
Sequence “logo” plots of the 19 residues (in bold) within the Alphacoronavirus‐1 RBD region known to interact with the receptor and the additional 11 residues detected to be under episodic positive selection (indicated with red plus signs). (a) A logo based on the 10 CCoVII S gene sequences obtained in this study (eight variants from the spotted hyena and two from the silver‐backed jackal). (b) A logo based on 55 Alphacoronavirus‐1 sequences including TGEV, PRCV, CCoVII, CCoVIIb and FCoVII (Table S4). The reference porcine Alphacoronavirus‐1 PRCV sequence is shown between the plots. In bold and underlined are the residues that directly interact with the host APN. The four residues indicated with yellow triangles are considered center residues for binding of the virus to its host receptor and these were identical in all variants. Red crosses indicate sites under positive selection. The overall height of each letter is proportional to sequence conservation as measured in bits. In each position the residue letters are ordered from the most to the least frequent. Polar residues are in black, acidic residues are in blue, basic residues are in green and nonpolar residues are in red. The sequence logos were drawn using software weblogo (weblogo.berkeley.edu)
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