Targeting the YXXΦ Motifs of the SARS Coronaviruses 1 and 2 ORF3a Peptides by In Silico Analysis to Predict Novel Virus-Host Interactions

Abstract

The emerging SARS-CoV and SARS-CoV-2 belong to the family of "common cold" RNA coronaviruses, and they are responsible for the 2003 epidemic and the current pandemic with over 6.3 M deaths worldwide. The ORF3a gene is conserved in both viruses and codes for the accessory protein ORF3a, with unclear functions, possibly related to viral virulence and pathogenesis. The tyrosine-based YXXΦ motif (Φ: bulky hydrophobic residue-L/I/M/V/F) was originally discovered to mediate clathrin-dependent endocytosis of membrane-spanning proteins. Many viruses employ the YXXΦ motif to achieve efficient receptor-guided internalisation in host cells, maintain the structural integrity of their capsids and enhance viral replication. Importantly, this motif has been recently identified on the ORF3a proteins of SARS-CoV and SARS-CoV-2. Given that the ORF3a aa sequence is not fully conserved between the two SARS viruses, we aimed to map in silico structural differences and putative sequence-driven alterations of regulatory elements within and adjacently to the YXXΦ motifs that could predict variations in ORF3a functions. Using robust bioinformatics tools, we investigated the presence of relevant post-translational modifications and the YXXΦ motif involvement in protein-protein interactions. Our study suggests that the predicted YXXΦ-related features may confer specific-yet to be discovered-functions to ORF3a proteins, significant to the new virus and related to enhanced propagation, host immune regulation and virulence.

Keywords: ORF3a; SARS-CoV; SARS-CoV-2; YXXΦ motif; immune response; post-translational modifications.

Figure 1

Alignment of MERS-CoV-ORF3, SARS-CoV-ORF3a and SARS-CoV-2-ORF3a sequences. YΧΧΦ motifs are represented in yellow and the YXXΦ-like tetrapeptides at the same position, in grey. Stars: amino acid sequence similarity; colons: amino acid sequence difference.

Figure 2

Topology of SARS-CoV-ORF3a and SARS-CoV-2-ORF3a YXXΦ motifs. (a) Schematic representation of the location of YXXΦ motifs and YXXΦ–like tetrapeptides within the SARS-CoV-ORF3a and SARS-CoV-2-ORF3a proteins is shown in yellow and grey, respectively. The non-canonical YXXXΦ motifs, the reverse ΦXXY motifs and the di-leucine motifs are represented in black frames, with black arrows and in light orange frames, respectively. Missing amino acids are denoted by a dash. (b) Schematic representation of the TM regions of SARS-CoV-ORF3a and SARS-CoV-2-ORF3a and indication of the topology of canonical ΥΧΧΦ motifs and ΥΧΧΦ-like tetrapeptides. YΧΧΦ motifs are presented in yellow circles and ΥΧΧΦ-like tetrapeptides, in grey circles. Extra: Extracellular; Intra: Intracellular.

Figure 3

Structural prediction analysis of SARS-CoV-ORF3a and SARS-CoV-2-ORF3a proteins. (a) Schematic representation of secondary structure of SARS-CoV-ORF3a and SARS-CoV-2-ORF3a proteins. Red boxes represent the α-helix structures, green boxes the β-sheet structures and gray boxes depict coil segments. (b) Prediction of the internal disordered regions in SARS-CoV-ORF3a and SARS-CoV2-ORF3a proteins. The disordered residues are noted with the letter “D”. YXXΦ motifs are presented in yellow and the YXXΦ-like tetrapeptides in grey.

Figure 4

Prediction of putative phosphorylated residues within the YXXΦ motifs, the YXXΦ-like tetrapeptides and their adjacent sequences in SARS-CoV-ORF3a and SARS-CoV-2-ORF3a proteins. The YXXΦ motifs are shown in yellow and the YXXΦ-like tetrapeptides in grey. P: Phosphorylation.

Figure 5

Prediction of a putative ubiquitination PTM on a SARS-CoV2-ORF3a YXXΦ-like tetrapeptide (in grey). The corresponding YXXΦ motif in SARS-CoV-ORF3a is depicted in yellow. The residues indicated in black circles promote Lys ubiquitination. Ub: Ubiquitination.

Figure 6

Prediction of a putative succinylation PTM on a SARS-CoV-ORF3a YXXΦ motif (in yellow). The residues indicated in black circles promote Lys succinylation. The residues in red circles inhibit Lys succinylation of the YXXΦ–like tetrapeptide of SARS-CoV-2-ORF3a protein (in grey). Succ: Succinylation.

Figure 7

Prediction of putative Lys methylation on (a) Lys75 and (b) on Lys235 of SARS-CoV-ORF3a and SARS-CoV-2-ORF3a YXXΦ motifs (in yellow) and YXXΦ-like tetrapeptides (in grey). Missing amino acids are denoted by a dash. Met: Methylation.

Figure 8

Prediction of an acetylation PTM in a YXXΦ-like tetrapeptide (in grey) of SARS-CoV-2-ORF3a. The corresponding YXXΦ motif of SARS-CoV-ORF3a is shown in yellow. Black circled residues are predicted to promote acetylation. Ac: Acetylation.

Figure 9

Prediction of (a) putative Tyr nitration and (b) an S-nitrosylation event on SARS-CoV-ORF3a and SARS-CoV-2-ORF3a YXXΦ motifs (in yellow). Grey denotes a YXXΦ-like tetrapeptide. The residues indicated in black circles promote Tyr and Cys PTMs, while the red circles have the opposite effect.

Figure 10

Prediction of atypical N-glycosylation PTMs (denoted by an open box) on the third SARS-CoV-ORF3a and SARS-CoV-2-ORF3 YXXΦ motif (in yellow). NLG: N-linked glycosylation.

Figure 11

Prediction of a putative S-glutathionylation on SARS-CoV-2-ORF3a CVVL tetrapeptide (in grey). The residues indicated in black circles promote Cys glutathionylation, while the residues in red circles may inhibit it. The corresponding YVVV motif of SARS-CoV-ORF3a protein is shown in yellow. GSH: Glutathionylation.

Figure 12

Location of putative N-myristoylation sites in SARS-CoV-ORF3a and SARS-CoV-2-ORF3a proteins. Residues noted with boxes belong to the myristoylation motif and partially overlap or are found adjacently to YXXΦ motifs (in yellow) and YXXΦ-like tetrapeptides (in grey). NMT: N-Myristoylation.

Figure 13

Prediction of putative sulfation PTMs on SARS-CoV-2-ORF3a YXXΦ motifs (in yellow). The residues indicated in black circles or boxes promote sulfation, while the residues in the open red box could inhibit it. YXXΦ–like tetrapeptides of both proteins are depicted in grey.

Figure 14

(a) Three-dimensional structures of SARS-CoV-ORF3a (structure on the left) and SARS-CoV-2-ORF3a (structure on the right) dimers. Residues of the YXXΦ motifs are shown as sticks and indicated by arrows. In the case of the fourth conserved motif, the SCoV-extra and the S2CoV-downstream motifs, the arrows point at the Tyr residue of the tetrapeptides. The carbon atoms of the motifs where Tyr is located inside the dimer’s interface are coloured in purple, while the ones facing the exterior are coloured in cyan. (b) Superimposition of the SARS-CoV-ORF3a dimer with that of the SARS-CoV-2-ORF3a one, focusing on the areas of the alternative conduction pathway (see reference [50] for a definition of the pathway). The figures were prepared with VMD 1.9.3.

Figure 15

(a) Prediction of linear B-cell epitopes, on SARS-CoV-ORF3a and SARS-CoV-2-ORF3a ΥΧΧΦ motifs and ΥΧΧΦ-like tetrapeptides, using the Chou & Fasman method of Beta-turn prediction. Residues with scores above the threshold (0.95) are coloured in yellow and the ones below the threshold in green. (b) Prediction of a conserved ITIM motif overlapping the 3rd YXXΦ motif. Positions of YXXΦ motifs and YXXΦ-like tetrapeptides, are noted in red and black, respectively.