Fig. 4.

Palmitoylation of TGEV S is dispensable for interaction with the M protein. (A) Schematic illustration of TGEV S_Y/A and TGEV S cysteine-mutants with Y/A-mutations. The cysteine-rich motif (CRM) and the destroyed tyrosine-based retention signal are highlighted. Substitutions by alanine are written in bold. ED, ectodomain; TMD, transmembrane domain; CD, cytoplasmic domain. (B) Every TGEV S cysteine-mutant shows an intracellular clustering in co-expression with the HA-tagged TGEV M protein. BHK-21 cells expressing either the S proteins alone (mutants shown in A, S wt, or VSV G) or in co-expression with the TGEV M protein were permeabilized with Triton-X 100 prior to antibody incubation. The same field is shown in each set of images for the coexpression of S and M. (C) Every TGEV S cysteine-mutant is retained intracellularly when co-expressed with the HA-tagged TGEV M protein. The S protein (or the VSV G protein) was detected on the cell surface of non-permeabilized BHK-21 cells expressing either the S proteins alone or in co-expression with the TGEV M. The detection of the HA-tagged TGEV M protein was done after permeabilization with Triton-X 100 prior to antibody incubation. The same field is shown for the detection of surface-expressed S together with the total M staining. (D) Comparison of the role of coronaviral S protein palmitoylation for MHV (references indicated), SARS-CoV (references indicated), and TGEV (this study) for S–M protein interaction and S incorporation into VLPs or virions.