A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport

Abstract

The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone's response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson's coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187-188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson's coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II β-turn between the anti-parallel β4 and β5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.

Keywords: Golgi apparatus; ORF3a; SARS-CoV-2; diG motif; diG-YXXΦ interaction; intracellular transport; lysosome; mutagenesis.

FIGURE 1

SARS-CoV-2 ORF3a protein localizes predominantly on the membranes of lysosomes and late endosomes. (A) Expression of ORF3a in the A549 cells showed abundant presence of ORF3a in late endosomes (P = 0.76 ± 0.04) as labeled by late endosome-specific marker Rab7 (a) and lysosomes (P = 0.77 ± 0.05) by LAMP-1 (b), with minor presence in mitochondria (P = 0.16 ± 0.02) by CoxIV (c), Golgi apparatus (P = 0.20 ± 0.04) by Giantin (d), and ER (P = 0.37 ± 0.04) by SEC61 (e). Enlarged image inserts in (A) and (b) show predominant association of ORF3a on the membranes of lysosomes and late endosomes. A FLAG-tagged ORF3a-carrying plasmid was transfected into A549 cells for 24 h post-transfection (hpt) before cells were collected for imaging. Collected cells were fixed and stained for ORF3a with anti-FLAG in green. All organelles were detected by Texas red-labeled secondary antibody in red except the ER, which was co-transfected with a pSEC61-RFP plasmid (e). Nuclei were detected by DAPI straining (blue). (B) Predominant localization of ORF3a in late endosomes (a) and lysosomes (b) in SARS-CoV-2 infected A549-hACE2 cells. A SARS-CoV-2 strain (USA-WA1/2020) was used to infect A549-hACE2 cells at an MOI of 0.5 for 48 h. Infected cells were fixed for IFA using anti-ORF3a (green), anti-Rab7 (a, red) and anti-LAMP1(b, red). Scale bar = 10 µm. Similar results were also seen in a different lung epithelial Calu-3 cell line (Supplementary Figure S1A). (C) and (D) Quantification of co-localization of ORF3a with different organelles as shown in (A) and (B), respectively. To quantify the co-localization of ORF3a (green) and different organelles (red), we used ImageJ2 and JACoP plugin to analyze ORF3a co-localization with proteins of different organelles (See Materials and Methods). Both the Pearson’s correlation coefficients (P) and Mander’s overlap coefficients (M) were obtained. A total of 50 random images were used for the quantitation and the calculation of the mean and standard deviation of the P- and M-values.

FIGURE 2

Mutagenesis of putative ORF3a functional and structural domains and corresponding changes of subcellular locations. (A) Schematic diagram showing where the deletions and single aa mutations were made. dDM1—dDM7 are deletions of putative functional domain motifs based on previous SARS-CoV and SARS-CoV-2 studies (Issa et al., 2020; Zhang et al., 2022b). dCR1 - dCR4, are deletions of presumably structural important cytoplasmic regions (CR) based on the bioinformatic analysis (this study). (B) Only those ORF3a mutants that showed different subcellular localizations from the WT are shown here. Those ORF3a mutants that show similar phenotypes to the WT are listed in Supplementary Figure S2. (C) ORF3a mutants show shift of subcellular localization from lysosomes to Golgi. A gene expression plasmid that carries a HA-tagged ORF3a at its C-terminal was co-transfected into A549 cells with a pRFP-LAMP1 plasmid. Transfected cells were collected 24 hpt and were fixed for the IFA to stain ORF3a using anti-HA antibody as shown in green. RFP, red fluorescent protein. Golgi apparatus was detected using anti-Giantin antibody as shown in red. Scale bar = 10 µm. (D,E) Quantification of co-localization of WT or mutated ORF3a with lysosome (D) or Golgi apparatus (E). A total of 50 random images were used for the quantitation and the calculation of the mean and standard deviation of the P- and M-values. A pair-wise students t-test was used to evaluate possible statistical difference between the WT and a mutant ORF3a at the levels of significance ****p < 0.0001

FIGURE 3

Interruption of any diG residues of ORF3a results in significant Golgi retention like deletion of the YXXΦ motif. Four diG-related ORF3a mutants were tested with the WT ORF3a and an YXXΦ motif mutant (dDM5) as controls. The four diG-related mutants include a deletion mutant of dCR1 (d175-194) that spans the diG motif and deletes the C-terminal end of the β4 sheet and N-terminal end of the β5 sheet, deletions of a single G residue (dG188) and both residues (dG187/dG188) as well as a single aa transition from glycine to tyrosine at the residue of 188 (G188Y). (A) Showing diminished association of the diG-related and YXXΦ motif mutant ORF3a from lysosomes, that were shown in red by anti-LAMP-1. (B) Showing the same diG-related and the YXXΦ motif mutations as in (A) resulted in increased presence in the Golgi apparatus. The Golgi apparatus was detected using anti-Giantin antibody as shown in red. Quantification of co-localization of WT or mutated ORF3a with lysosome (C) or Golgi apparatus (D) by using the same method as described in Figure 2.

FIGURE 4

The dG188 mutant loses its ability to move out of the Golgi apparatus. The WT ORF3a or the dG188 mutant-carrying plasmid was transfected into A549 cells for 5 h. The cell cultures transfected with either plasmid was then treated with or without 1 µM Brefeldin A (BFA). Twenty hours after BFA treatment, the cells were fixed for the IFA using anti-HA antibody to stain ORF3a (green). The LAMP-1 antibody (red) was used to show lysosomes (A), and the Giantin antibody (red) was used to show the Golgi apparatus (B). Scale bar: 10 µm.

FIGURE 5

Predicated interaction of the diG motif with the YXXΦ motif. (A) Four residues, I186, diG motif (G187 and G188), and Y189, form a type II β-turn between β4 and β5 sheets. The hydrogen bonds were indicated with yellow dash lines. (B) The diG motif (G187 and G188) interacts with N161 and S162 residues of the YXXΦ motif of opposite monomer. (C) The diG motif and the YXXΦ motif from opposite monomers in the dimer forms a “hand-in-hand” shape structure. Only the aa 159–164 (PYNSVT) and 185–189 (QIGGY) containing YXXΦ and diG motifs (underlined) are shown. (D) Predicted protein structures of diG motif mutations (top panel) and their alignments with WT ORF3a (bottom panel). Red arrows indicate the major differences between WT and mutants. The protein structure was predicted using the SWISSMODEL program. 7KJR was used as the template. (E) The prominent aromatic side chain of Y188 blocks the formation of the “hand-in-hand” structure. All the protein 3D structures were visualized with PyMOL. All the images were prepared using Adobe Illustrator 2020.