Fig. 2.

Palmitoylation of TGEV. (A) Treatment with 2-bromopalmitate (2-BP) decreases the infectivity of TGEV. ST or BHK-21 cells were inoculated with TGEV or VSV, respectively. At 1 h p.i., media were replaced with growth media containing the indicated concentrations of 2-BP or ethanol as 2-BP solvent (ethanol control: amount of ethanol present in the highest 2-BP concentration; 0.0 µM 2-BP: medium without ethanol). Twenty-four hours later, media were collected and the amount of infectious virus particles was determined by plaque assay. Plaque forming units (PFU)/ml are displayed as mean+standard deviation (SD). ^⁎, significantly different according to Student׳s t test compared to 0.0 µM 2-BP (p<0.05). (B) 2-BP is not cytotoxic to ST cells at the concentrations used. ST cells were treated with the indicated concentrations of 2-BP and cell proliferation was determined by WST 1-assay. The cell proliferation is indicated as mean+SD in percentage. (C) The amount of viral proteins present in viral particles is reduced after 2-BP treatment. Virions from cell culture supernatants of infected cells, treated with the indicated amounts of 2-BP, were pelleted by ultracentrifugation 24 h p.i. and analyzed by SDS-PAGE and Western blotting (virions). The indicated proteins (S, N, and M) were detected by monoclonal antibodies. The corresponding cells were lysed and the respective proteins were equally analyzed (cell lysates). (D) Palmitoylation of the TGEV S protein in virus particles. Infected and mock-infected ST cells were labeled with [³H]-palmitic acid or [³⁵S]-methionine/cysteine. Radiolabeled virus was analyzed by SDS-PAGE and fluorography. A hydroxylamine treatment was performed to determine the linkage of fatty acids via a thioester bond.