A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

NIZZTI33 was produced using both N122 and T133 oligonucleotides as Figure 2 . Gml is retained in the Golgi region of transfected cells . COS cells expressing either G or Gml were fixed and stained by doublelabel indirect immunofluorescence microscopy. Surface G protein was detected by staining with rabbit antiVSV serum followed by a Texas red-conjugated second antibody. Internal G protein was detected after permeabilization with a monoclonal anti-G antibody and a fluorescein-conjugated second antibody. Left panels were photographed with the fluorescein filter, and those on the right are the same field photographed with the rhodamine filter. Bar, 10 /Am . primers for second strand synthesis . These same oligonucleotides were used to create the mutations in the El deletion mutant, Gm2,3 . Gmlins was produced with the mlins oligonucleotide, but Gm1QI was obtained only after a longer oligonucleotide, 5'-CTTAACCATAATACTTATCTATGGCTATGCAACCC-3' was used . T4 DNA polymerase (Biolabs) was used for second strand synthesis, and the double-stranded molecules were transfected into E. coli NM522 . Single-stranded DNA from three to six plaques was sequenced using the dideoxy procedure (Sequenase, USB) to select the desired mutations. The mutated genes were excised from the double-stranded replicadve form DNA and subcloned into both the SV-40 expression vector pJC119 (44) and the T7 expression vector pAR2529 (10) . All general recombinant DNA techniques were as described (41) .