VIROLOGY 109, 165-173 (1981) Reversible Defect in the Glycosylation of the Membrane Proteins of Semliki Forest Virus ts-1 Mutant MARJA PESONEN,’ JAAKKO SARASTE, KATSUYUKI HASHIMOTO, AND LEEVI KAARIAINEN Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 2.9, Finla& Accepted September 12, 1980 Here we have characterized the oligosaccharide chains ofthe viral membrane proteins from chicken embryo fibroblasts infected with tsl mutant of Semliki Forest virus. When the in- fected cells were labeled with [:‘H]mannose and maintained at 39”, the radioactive oligosac- charides were exclusively of the high mannose type as evidenced by their sensitivity to endo- glycosidase H and their high affinity to immobilized concanavalin A. When the infected cells were labeled at 39” followed by chase at 28” in the presence of cycloheximide, about 35% of the high mannose type oligosaccharides were converted to complex oligosaccharides as evi- denced by their elution behavior from concanavalin A-Sepharose, resistance to endoglycosi- dase H, and sensitivity to mild acid hydrolysis. The rest of the oligosaccharides remained as high mannose type chains. The results suggest that at the restrictive temperature the viral membrane glycoproteins in Is-l infected cells are arrested in the rough endoplasmic reticu- lum, but start to be transported to the Golgi complex, once the cultures are shifted to the per- missive temperature. INTRODUCTION The envelope glycoproteins of Semliki Forest virus (SFV) and Sindbis virus are synthesized as a polyprotein from the sub- genomic 26 S mRNA (for reviews see Strauss and Strauss, 1977 and Kaariainen and Soderlund, 1978). After the N-terminal capsid protein has been cleaved from the growing polyprotein a signal sequence is revealed, which conducts the nascent ~62 (precursor of E2 and E3 of SFV) and PE2 (precursor of E2 of Sindbis virus) to the cisternal side of the rough endoplasmic re- ticulum (RE R) membrane (Wirth et al., 19’77; Garoffet al., 1978; Bonatti and Blobel, 1979; Bonatti et al., 1979). Once ~62 has been com- pleted, another signal sequence is presum- ably revealed, which leads the N-terminus of the C-terminal El protein through the RER membrane (Hashimoto et al., unpub- lished; K. Simons, personal communication). El and p62 (PE2) attain their oligosaccha- ride chains probably during their synthesis at the RER membrane (Garoff et al., 1978; Garoff and Schwartz, 1978; Bonatti et al., 1979) from a dolicholpyrophosphate-oligo- saccharide intermediate (Krag and Robbins, 1977; Leavitt et al., 1977; Sefton, 1977; Schwartz et al., 1978). The envelope proteins of SFV and Sindbis virus have complex oligosaccharide chains with sialosyl-N-acetyllactosamine branches and fucose attached to the core pentasaccha- ride Mancul-3(Mancul-6)Man~l-4GlcNac~l- 4GlcNac” (Pesonen, 1979; Pesonen et al., 1979; Burke and Keegstra, 1979) as well as high mannose type oligosaccharides with two to four cu-mannose residues linked to the same core pentasaccharide (Mattila and Renkonen, 1978; Burke and Keegstra, 1979). We have recently shown that certain tem- perature-sensitive mutants of SFV and Sindbis virus have a reversible defect in the transport of envelope glycoproteins. At the restrictive temperature (39”) the glycopro- teins are not found at the cell surface but migrate there when the cultures are shifted to the permissive temperature (2sO) in the ’ To whom reprint requests should be addressed. Z GlcNAc, A’-acetylglucosamine; Man, mannose. 165 0042-6822/81/030165-09$02.00/O Copyright F 1981 by Academic Press. Inc. All rights of reproductinn m any form rrwrvrd