Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

Abstract

Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

Fig. 1

SARS-CoV S palmitoylation can occur in a pre-medial Golgi compartment and is a stable modification. (A) At 24 h post-transfection, HEK293T cells expressing SARS-CoV S were labeled with ³⁵S-methionine/cysteine or ³H-palmitic acid for 30 min. After lysis, S protein was immunoprecipitated, denatured, digested with endo H, separated by SDS–PAGE and imaged by fluorography. (B) At 24 h post-transfection, HEK293T cells expressing SARS-CoV S were labeled with ³⁵S-methionine/cysteine or ³H-palmitic acid for 30 min and chased for 0, 40, 80 or 120 min. After lysis, S protein was immunoprecipitated, separated by SDS–PAGE and analyzed by fluorography for 24 h (³⁵S) or for 1 wk (³H).

Fig. 2

SARS-CoV S lacking all cytoplasmic cysteines is not palmitoylated. (A) Cytoplasmic tail sequence of SARS-CoV S and a mutant lacking all 9 cytoplasmic cysteines (SARS-CoV S_PN). (B) At 24 h post-transfection, HEK293T cells expressing SARS-CoV S or S_PN were labeled with ³⁵S-methionine/cysteine or ³H-palmitic acid for 30 min. After lysis, S protein was immunoprecipitated, separated by SDS–PAGE and analyzed by fluorography. Longer exposure (6 weeks) of the ³H-palmitate labeled samples confirmed the absence of palmitoylated SARS-CoV S_PN.

Fig. 3

SARS-CoV S and SARS-CoV S_PN have similar half-lives. At 24 h post-transfection, HEK293T cells expressing SARS-CoV S (solid line) or S_PN (dashed line) were labeled with ³⁵S-methionine/cysteine for 20 min and chased for 0, 1, 2 or 3 h. After lysis, S protein was immunoprecipitated, separated by SDS–PAGE and analyzed by autoradiography. Percentage of S or S_PN remaining at each time point was calculated using the amount of S or S_PN at 0 h chase as 100%. The average of 3 independent experiments ± SEM is shown.

Fig. 4

SARS-CoV S and SARS-CoV S_PN both localize to the cell surface. (A) At 24 h post-transfection, HEK293T expressing SARS-CoV S or S_PN were fixed, permeabilized and co-stained with mouse anti-SARS-CoV S and rabbit anti-golgin 160 (a Golgi marker). (B) At 24 h post-transfection, unpermeabilized HEK293T cells were stained with mouse anti-SARS-CoV S at 0 °C for 20 min to label SARS-CoV S or S_PN present on the cell surface. Cells were then fixed, permeabilized and co-stained with rabbit anti-SARS-CoV S to label total S protein. (A and B) Secondary antibodies were Alexa 488-conjugated donkey anti-mouse IgG and Texas Red-conjugated donkey anti-rabbit IgG. The same field is shown in each set of images. (C) At 24 h post-transfection, SARS-CoV S or S_PN present at the plasma membrane was biotinylated with a membrane-impermeable biotinylating agent. After lysis, biotinylated S proteins were recovered with streptavidin agarose and analyzed by SDS–PAGE and Western blotting with rabbit anti-SARS-CoV S. The graph shows quantification from 3 independent experiments ± SEM.

Fig. 5

SARS-CoV S and SARS-CoV S_PN can be retained at the Golgi by SARS-CoV M. (A) At 24 h post-transfection, HEK293T cells expressing SARS-CoV S or S_PN and SARS-CoV M were fixed, permeabilized and co-stained with mouse anti-SARS-CoV and rabbit anti-SARS-CoV M. Secondary antibodies were Alexa 488-conjugated donkey anti-mouse IgG and Texas Red-conjugated donkey anti-rabbit IgG. (B) At 24 h post-transfection, HEK293T cells expressing SARS-CoV S or SARS-CoV S_PN were labeled with ³⁵S-methionine/cysteine for 20 min and chased for 0, 20 or 40 min. After lysis, S protein was immunoprecipitated, denatured, digested with endo H, separated by SDS–PAGE and analyzed by autoradiography. The average of 3 independent experiments ± SEM is shown.

Fig. 6

SARS-CoV S palmitoylation is necessary for partitioning into detergent-resistant membranes. At 24 h post-transfection, detergent-resistant membranes (DRMs) were extracted from HEK293T cells expressing SARS-CoV S or S_PN using cold Triton. DRMs were isolated using discontinuous density ultracentrifugation, and fractions were collected from the top. S protein was identified by Western blotting (upper and middle panels) and DRMs were identified using HRP-cholera toxin B, which binds ganglioside GM1 (bottom panel). A representative image of 3 independent experiments is shown.

Fig. 7

SARS-CoV S palmitoylation is necessary for cell–cell fusion. At 48 h post-transfection, Vero cells expressing SARS-CoV S (A) or SARS-CoV S_PN (B) were trypsinized then re-plated. At 24 h post-trypsinization, the number of nuclei per syncytia was counted; the average of 3 independent experiments ± SEM is shown (C).