The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction

Abstract

Virus-like particle (VLP) formation by the coronavirus E and M proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. We studied interactions between the infectious bronchitis virus (IBV) E and M proteins using in vivo crosslinking and VLP assembly assays. We show that IBV E and M can be crosslinked to each other in IBV-infected and transfected cells, indicating that they interact. The cytoplasmic tails of both proteins are important for this interaction. We also examined the ability of the mutant and chimeric E and M proteins to form VLPs. IBV M proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support VLP formation, regardless of their ability to be crosslinked to IBV E. Interactions between the E and M proteins and the membrane bilayer are likely to play an important role in VLP formation and virus budding.

Fig. 1

IBV E and M colocalize and interact in IBV-infected Vero cells. (A) IBV-infected Vero cells were fixed for immunofluorescence at 6 h postinfection and double labeled with rat anti-E antibody (a) and rabbit anti-M antibody (b). Secondary antibodies were fluorescein-conjugated goat anti-rat IgG and Texas red conjugated donkey anti-rabbit IgG. Panel c is a differential interference contrast (DIC) image of the labeled cells. Bar, 15 μm. (B) IBV-infected Vero cells were labeled with [³⁵S]methionine-cysteine at 45 h postinfection, treated with DSP as indicated, lysed, and immunoprecipitated with anti-E or anti-M antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–15% PAGE, in the presence or absence of β-mercaptoethanol (βME) as indicated, and fluorography. IBV E is coprecipitated with IBV M after crosslinking and vice versa. These data are representative of at least three independent experiments.

Fig. 2

IBV E and M interact in transfected OST7-1 cells. OST7-1 cells expressing E and M proteins alone or together were labeled with [³⁵S]methionine-cysteine, treated with DSP as indicated, lysed, and immunoprecipitated with anti-E or anti-M antibodies as described under Materials and methods. All immunoprecipitates in this and the following figures were deglycosylated by treating with N-glycanase to collapse the M protein to a single band. The immunoprecipitates were analyzed by SDS–17.5% PAGE in the presence of βME, and fluorography. The asterisk indicates a band that corresponds to M protein dimers. These data are representative of at least three independent experiments.

Fig. 3

The wild-type and mutant versions of IBV E and IBV M proteins used in this study are localized to the Golgi region of transfected OST7-1 cells. (A) OST7-1 cells expressing wild-type IBV E and M proteins (a–c), EG3 and wild-type M protein (d–f), CTE and wild-type M protein (g–i), or GEt and wild-type M protein (j–l) were fixed for immunofluorescence and double labeled with rat anti-E antibody (a, d, g, and j) and rabbit anti-M antibody (b, e, h, and k). Secondary antibodies were fluorescein-conjugated goat anti-rat IgG and Texas red conjugated donkey anti-rabbit IgG. The third image in each row (c, f, i, and l) is a DIC image of the labeled cells. In the diagrams of the E mutant and chimeric proteins, IBV E sequence is shown in black and VSV G sequence is shown in gray. Bar, 10 μm. (B) OST7-1 cells expressing wild-type E protein and MctΔ1 (a–c), MctΔ2 (d–f), MctΔ3 (g–i), or MΔm2,3 (j–l) were fixed for immunofluorescence and double labeled with rat anti-E antibody (a, d, g, and j) and rabbit anti-M antibody (b, e, h, and k). Secondary antibodies were fluorescein-conjugated goat anti-rat IgG and Texas red conjugated donkey anti-rabbit IgG. The third image in each row (c, f, i, and l) is a DIC image of the labeled cells. In the diagrams of the deletion mutant M proteins, the thin lines indicate deleted sequence. Bar, 10 μm.

Fig. 4

The cytoplasmic tail of IBV E is sufficient for interaction with IBV M. (A) OST7-1 cells expressing wild-type M protein and wild-type E protein or the E transmembrane replacement mutant proteins EG1, EG2, or EG3 were labeled with [³⁵S]methionine-cysteine, treated with DSP, lysed, and immunoprecipitated with anti-E or anti-M antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–17.5% PAGE in the presence of βME, and fluorography. The asterisk indicates a band that corresponds to M protein dimers. (B) OST7-1 cells coexpressing the GEt chimera and wild-type M protein were labeled with [³⁵S]methionine-cysteine, treated with DSP, lysed, and immunoprecipitated with anti-E or anti-M antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–15% PAGE in the presence of βME, and fluorography. The asterisk indicates a band that corresponds to M protein dimers. (C) OST7-1 expressing CTE protein alone or with M protein were labeled with [³⁵S]methionine-cysteine, treated with DSP, lysed, and immunoprecipitated with anti-E antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–17.5% PAGE in the presence of βME, and fluorography. These data are representative of at least three independent experiments.

Fig. 5

A region of the IBV M cytoplasmic tail is required for crosslinking to IBV E. OST7-1 cells expressing wild-type E protein and wild-type M protein or the M deletion mutant proteins MctΔ1, MctΔ2, MctΔ3, or MΔm2,3 were labeled with [³⁵S]methionine-cysteine, treated with DSP, lysed, and immunoprecipitated with anti-E or anti-M antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–17.5% PAGE in the presence of βME, and fluorography. The asterisks indicate bands that correspond to M, MctΔ3, or MΔm2,3 protein dimers. These data are representative of at least three independent experiments.

Fig. 6

Complete replacement of the E transmembrane domain does not affect VLP formation. OST7-1 cells expressing the indicated proteins were labeled with [³⁵S]methionine-cysteine for 1 h and chased for 3 h, and the supernatants and cells were harvested and immunoprecipitated with anti-E and anti-M antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–17.5% PAGE and fluorography. The supernatant samples (lanes 12–22) were exposed to film 20 times longer than the cell samples (lanes 1–11) to allow the visualization of EG2 and EG3 proteins in lanes 18 and 20, respectively. The asterisk indicates a band that corresponds to dimers of M protein. These data are representative of at least three independent experiments.

Fig. 7

Cytoplasmic and transmembrane region deletion mutants of IBV M do not support VLP formation. OST7-1 cells expressing the indicated proteins were labeled with with [³⁵S]methionine-cysteine for 1 h and chased for 3 h, and the supernatants and cells were harvested and immunoprecipitated with anti-E and anti-M antibodies as described under Materials and methods. The immunoprecipitates were analyzed by SDS–17.5% PAGE and fluorography. The supernatant samples (lanes 12–22) were exposed to film 13 times longer than the cell samples (lanes 1–11). The MctΔ1 and MctΔ2 mutants are not radiolabeled as efficiently as wild-type M and the MctΔ3 and MΔm2,3 mutants because they have fewer methionines and cysteines due to their deletions. These data are representative of at least three independent experiments.