Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site

Abstract

Coronavirus budding at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) requires accumulation of the viral envelope proteins at this point in the secretory pathway. Here we demonstrate that the spike (S) protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a canonical dilysine endoplasmic reticulum retrieval signal (-KKXX-COOH) in its cytoplasmic tail. This signal can retain a chimeric reporter protein in the ERGIC and when mutated allows transport of the full-length S protein as well as the chimera to the plasma membrane. Interestingly, the IBV S protein also contains a tyrosine-based endocytosis signal in its cytoplasmic tail, suggesting that any S protein that escapes the ERGIC will be rapidly endocytosed when it reaches the plasma membrane. We also identified a novel dibasic motif (-KXHXX-COOH) in the cytoplasmic tails of S proteins from group 1 coronaviruses and from the newly identified coronavirus implicated in severe acute respiratory syndrome. This dibasic motif also retained a reporter protein in the ERGIC, similar to the dilysine motif in IBV S. The cytoplasmic tails of S proteins from group 2 coronaviruses lack an intracellular localization signal. The inherent differences in S-protein trafficking could point to interesting variations in pathogenesis of coronaviruses, since increased levels of surface S protein could promote syncytium formation and direct cell-to-cell spread of the infection.

FIG. 1.

Mutation of the C-terminal dilysine signal promotes transport of IBV S to the cell surface. (A) The sequence of the IBV S cytoplasmic tail, with the KKXX motif at the C terminus. Alanine residues (indicated with arrows) replace the dilysine motif in the S_2A mutant. A potential endocytosis signal (YYTF) is indicated in italics. (B) Intact HeLa cells expressing wild-type IBV S or S_2A were stained with mouse anti-IBV S at 4°C and then fixed, permeabilized, and stained with rabbit anti-IBV S for internal expression. Secondary antibodies were fluorescein-conjugated donkey anti-rabbit immunoglobulin G (IgG) and Texas Red-conjugated goat anti-mouse IgG. Bar, 10 μm.

FIG. 2.

The IBV S cytoplasmic tail retains a reporter protein intracellularly. (A) The G-S chimera contains the lumenal head (black) and transmembrane domain (gray) from VSV-G and the cytoplasmic domain from IBV S (white; sequence shown in Fig. 1A). G-S_2A has the dilysine signal mutagenized to alanines residues, and G-S_4A has both the upstream tyrosine internalization signal and the dilysine signal mutagenized to alanine residues. (B) Intact HeLa cells expressing VSV-G, G-S, G-S_2A, or G-S_4A were stained with mouse anti-VSV-G at 4°C, fixed, permeabilized, and then stained with rabbit anti-VSV-G for internal expression. Secondary antibodies were fluorescein-conjugated donkey anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. Bar, 10 μm.

FIG. 3.

G-S is retained mostly in the ERGIC, and mutation of the dilysine motif results in transport to the plasma membrane and endocytosis. (A) HeLa cells expressing G-S were fixed, permeabilized, stained with either rabbit or mouse anti-VSV-G, and double labeled with either rabbit anticalnexin, mouse anti-ERGIC-53, or mouse anti-GM130. For cells double labeled with calnexin, the secondary antibodies were fluorescein-conjugated donkey anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. For cells double labeled with ERGIC-53 or GM130, secondary antibodies were fluorescein-conjugated goat anti-mouse IgG and Texas Red-conjugated donkey rabbit IgG. (B) Intact HeLa cells expressing G-S_2A and G-S_4A were incubated with mouse anti-VSV-G at 37°C for 15 min, fixed, permeabilized, and then stained with rabbit anti-VSV-G for internal expression. Secondary antibodies were fluorescein-conjugated donkey anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. Bars, 10 μm.

FIG. 4.

G-S is rapidly trafficked through the Golgi after mutation of the dilysine signal. (A) HeLa cells expressing VSV-G, G-S, G-S_2A, or G-S_4A were pulse labeled with [³⁵S] methionine-cysteine for 15 min, chased for the times indicated, lysed, and immunoprecipitated with anti-VSV-G antibody. The immunoprecipitates were treated with endo H and subjected to SDS-PAGE. The endo H-resistant and -sensitive forms are indicated. (B) Quantitation of oligosaccharide processing rates. These data represent the averages from four independent experiments, with the error bars representing the standard deviations.

FIG. 5.

The C-terminal 11 amino acids of S proteins from group 2 coronaviruses lack intracellular localization signals. (A) The final 11 amino acids of G-S were swapped with those from the BCV or MHV-A59 S proteins (underlined). Note the lack of any apparent dibasic motif at the −3 and −4 or −5 position from the C terminus in the BCV and MHV sequences. (B) Intact HeLa cells expressing G-S_2A, G-S_BCV, or G-S_MHV were stained with mouse anti-VSV-G at 4°C, fixed, permeabilized, and then stained with rabbit anti-VSV-G for internal expression. Secondary antibodies were fluorescein-conjugated donkey anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. Bar, 10 μm. (C) HeLa cells expressing G-S_2A, G-S_BCV, or G-S_MHV were pulse labeled with [³⁵S]methionine-cysteine for 15 min, chased for 0, 15, 30, or 60 min, lysed, and immunoprecipitated with anti-VSV-G antibody. The immunoprecipitates were treated with endo H and subjected to SDS-PAGE. Endo H resistance was quantitated for three independent experiments (error bars represent standard deviations).

FIG. 6.

Group 1 coronaviruses and SARS S proteins contain intracellular localization signals in their cytoplasmic tails. (A) The final 11 amino acids of TGEV S or SARS S (underlined), which contain putative dibasic signals, were swapped for the same residues of G-S. (B) Intact HeLa cells expressing G-S_SARS or G-S_TGEV were stained with mouse anti-VSV-G at 4°C, fixed, permeabilized, and then stained with rabbit anti-VSV-G for internal expression. Secondary antibodies were fluorescein-conjugated donkey anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. (C) HeLa cells expressing G-S, G-S_SARS, or G-S_TGEV were fixed, permeabilized, and stained with rabbit anti-VSV-G and mouse anti-ERGIC-53. Secondary antibodies were fluorescein-conjugated goat anti-rabbit IgG and Texas Red-conjugated donkey anti-rabbit IgG. The insets show enlargements of the boxed regions, with the anti-VSV-G panel on top and the anti-ERGIC-53 panel below. Bars, 10 μm.

FIG. 7.

Mutagenesis of the histidine and lysine residues in G-S_SARS and G-S_TGEV results in transport to the plasma membrane. (A) The histidine at −3 and the lysine at −5 were replaced by alanines (G-S_SARS2A and G-S_TGEV2A). (B) Intact HeLa cells expressing G-S_2A, G-S_SARS2A, or G-S_TGEV2A were stained with mouse anti-VSV-G at 4°C, fixed, permeabilized, and then stained with rabbit anti-VSV-G for internal expression. Secondary antibodies were fluorescein-conjugated donkey anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. Bar, 10 μm.

FIG. 8.

Mutation of the histidine and lysine residues in G-S_SARS and G-S_TGEV results in rapid trafficking through the Golgi complex. HeLa cells expressing G-S_SARS, G-S_TGEV, G-S_SARS2A, or G-S_TGEV2A were pulse labeled with [³⁵S]methionine-cysteine for 15 min, chased for 0, 15, 30, or 60 min, lysed, and immunoprecipitated with anti-VSV-G antibody. The immunoprecipitates were treated with endo H and subjected to SDS-PAGE. Endo H resistance was quantitated (three independent experiments, with the error bars representing the standard deviations).