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Abstract

Coronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all of the other structural proteins. Coronaviruses bud into the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), but the mechanisms by which M proteins are transported from their site of synthesis, the ER, to the budding site remain poorly understood. Here, we investigated the intracellular trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M protein. Subcellular localization analyses revealed that the MERS-CoV M protein is retained intracellularly in the trans-Golgi network (TGN), and we identified two motifs in the distal part of the C-terminal domain as being important for this specific localization. We identified the first motif as a functional diacidic DxE ER export signal, because substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, ¹⁹⁹KxGxYR²⁰⁴, was responsible for retaining the M protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage toward the plasma membrane. We further confirmed the role of ¹⁹⁹KxGxYR²⁰⁴ as a TGN retention signal by using chimeras between MERS-CoV M and the M protein of infectious bronchitis virus (IBV). Our results indicated that the C-terminal domains of both proteins determine their specific localization, namely TGN and ERGIC/cis-Golgi for MERS-M and IBV-M, respectively. Our findings indicate that MERS-CoV M protein localizes to the TGN because of the combined presence of an ER export signal and a TGN retention motif.

Keywords: MERS-CoV; Middle East respiratory syndrome; coronavirus; endoplasmic reticulum (ER); intracellular trafficking; plasma membrane; protein motif; protein sorting; trans-Golgi network localization; viral protein.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

👁 Figure 1.

Figure 1.

A, schematic drawing of the MERS-M protein with the sequence of residues 149–219 of the C-terminal domain. B and C, subcellular localization of the MERS-CoV M protein. Cells expressing HA-tagged M protein in combination with the GFP-CI-MPR, ERGIC-53-GFP, or CD4 fused with GFP were labeled with an anti-HA antibody. GFP-CI-MPR is a TGN marker, ERGIC-53-GFP is an ER–Golgi intermediate compartment marker, and CD4 is a protein expressed at the cell surface. To detect the ER compartment or the TGN, cells were double-labeled for HA and CRT or TGN46, as indicated. Bars, 20 μm. Pearson's correlation coefficients were calculated for each combination of co-staining. D and E, N-terminal or C-terminal added tags have no effect on the TGN localization of the M protein. Cells expressing untagged M protein (M), N- or C-terminally V5-tagged M protein (V5-M and M-V5 respectively), N-terminally HA-tagged M (HA-M), or C-terminally VSVG-tagged M (M-VSVG) were double-labeled with anti-M antibody together with an anti-TGN46 antibody. Pearson's correlation coefficients were calculated. Error bars, S.E.

👁 Figure 2.

Figure 2.

The distal part of MERS-M C-terminal domain contains motif(s) involved in its subcellular localization. Cells expressing the M protein (HA-M and M-VSVG) or the M protein lacking its last 20 residues (HA-MΔ20 or MΔ20-VSVG) or the M protein with Asp-211 and Glu-213 mutated into Ala (HA-M-DxE or M-DxE-VSVG) were processed for detection of M protein using an anti-tag antibody. The TGN was detected by using an anti-TGN46 (A), and the ER was detected by using an anti-CRT antibody (B). The plasma membrane was labeled by co-expression of CD4 fused to GFP together with the M protein (C). Bars, 20 μm. Pearson's correlation coefficients were calculated for each combination of co-staining (D). Error bars, S.E.

👁 Figure 3.

Figure 3.

Subcellular localization of mutants with serial deletions of the distal part of the MERS-CoV M protein. M protein with a C-terminal VSVG tag (M) or M protein deleted of 5 (MΔ5), 10 (MΔ10), 15 (MΔ15), or 20 amino acid residues (MΔ20) was expressed in HeLa cells, and its localization either in the TGN or at the plasma membrane was investigated by double labeling with TGN46 (A) or by co-expressing CD4 fused to GFP (B). Bars, 20 μm. Pearson's correlation coefficients were calculated for each combination of co-staining (C). Error bars, S.E.

👁 Figure 4.

Figure 4.

Identification of the MERS-CoV MΔ15 motif involved in its TGN localization. Amino acid residues Gly-201, Asn-202, Tyr-203, and Arg-204 located in the last 5 residues of MΔ15 (located in the TGN) were mutated individually into alanine, and the subcellular localization of the mutants was analyzed as described in the legend to Fig. 3. Bars, 20 μm. Error bars, S.E.

👁 Figure 5.

Figure 5.

The KxGxYR motif is involved in MERS-M protein localization in TGN. The mutations K199A, G201A, Y203A, and R204A were introduced individually or together in the context of the full-length M protein with a C-terminal VSVG tag. The subcellular localization of the mutants was analyzed as described in the legend to Fig. 3. Bars, 20 μm. Error bars, S.E.

👁 Figure 6.

Figure 6.

Cell surface expression of M protein mutants. Plasma membrane proteins of cells expressing the different M protein mutants were labeled with nonpermeable biotin. Biotinylated proteins were purified using streptavidin-conjugated agarose beads. Biotinylated M proteins and total M proteins in cell lysates were detected by immunoblotting (A) and quantified (B). Results are expressed as the percentage of total M protein expressed at the cell surface and are expressed as the mean of five independent experiments. Error bars, S.E. Results were analyzed by using an analysis of variance test (*, p < 0.1; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Glycosylation of M protein mutants is shown. Lysates of cells expressing the M protein or the M protein with the ER export signal mutated (M-DxE) or the TGN localization motif mutated (M-KGYR) were treated with EndoH or PNGase F. A lysate of cells expressing the M protein with its N-glycosylation site mutated (N3Q) was left untreated. N-terminal tagged proteins were detected by Western blotting with an anti-V5 antibody (C). Endocytosis of M protein and M-KGYR. Cells were transfected with vectors expressing the M and M-K199A-G210A-Y203A-R204A (M-KGYR) proteins. Then cell surface proteins were labeled with nonpermeable biotin at 4 °C. Endocytosis was allowed by incubating the cells for 30 min at 37 °C. Biotin of noninternalized proteins was cleaved with GSH. Internalized M protein was detected after purification with streptavidin-conjugated agarose beads by immunoblotting (E). In each experiment, each condition was performed in duplicate. For cell surface–associated protein, only 25% of the sample was loaded on the gel. For the controls of GSH cleavage (without any internalization, 0 min) and for the samples internalized (30 min), the totality of the samples were loaded on the gel. Internalized M protein was quantified. The results are expressed as the percentage of cell surface–associated M protein and are expressed as the mean of three independent experiments. Error bars, S.E. (E and F).

👁 Figure 7.

Figure 7.

MERS-M and IBV-M sequence alignment (A) and schematic drawings of the different IBV-M and MERS-M chimeras that were constructed (B). First, the C-terminal domain of MERS-CoV M was replaced with that of IBV-M (MERS-M/IBV-M), and the C-terminal domain of IBV-M was replaced by that of MERS-CoV M with or without the mutation of the KxGxYR motif (IBV-M/MERS-M-KGYR and IBV-M/MERS-M, respectively). The first membrane-spanning segment of MERS-CoV M was replaced with that of IBV-M in the context of the MERS-M-KGYR (TM1-IBV/MERS-M-KGYR), and the first membrane-spanning segment of IBV-M was replaced by that of MERS-CoV M (TM1-MERS/IBV-M). All of the chimeras were tagged at their C-terminal extremity with a VSVG epitope.

👁 Figure 8.

Figure 8.

Subcellular localization of IBV-M and MERS-CoV M proteins chimeras. The localization of the different chimeras in the TGN or in the ERGIC compartments was investigated by immunofluorescent double labeling by using an anti-VSVG antibody and an anti-TGN46 (A) or by expressing the ERGIC-53 marker conjugated with GFP (B). Bars, 20 μm. Pearson's correlation coefficients were calculated for each combination of co-staining (C). Error bars, S.E.

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References

1. Masters P. S. (2006) The molecular biology of coronaviruses. Adv. Virus Res. 66, 193–292 10.1016/S0065-3527(06)66005-3 - DOI - PMC - PubMed
1. Perlman S., and Netland J. (2009) Coronaviruses post-SARS: update on replication and pathogenesis. Nat. Rev. Microbiol. 7, 439–450 10.1038/nrmicro2147 - DOI - PMC - PubMed
1. Corman V. M., Muth D., Niemeyer D., and Drosten C. (2018) Hosts and sources of endemic human coronaviruses. Adv. Virus Res. 100, 163–188 10.1016/bs.aivir.2018.01.001 - DOI - PMC - PubMed
1. Belouzard S., Millet J. K., Licitra B. N., and Whittaker G. R. (2012) Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4, 1011–1033 10.3390/v4061011 - DOI - PMC - PubMed
1. Heald-Sargent T., and Gallagher T. (2012) Ready, set, fuse! The coronavirus spike protein and acquisition of fusion competence. Viruses 4, 557–580 10.3390/v4040557 - DOI - PMC - PubMed

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URL: https://pubmed.ncbi.nlm.nih.gov/31399512/

⇱ The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal - PubMed

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