key: cord-0004923-0ymcxcj3 authors: Repges-Illguth, S.; Kaluza, G. title: Differences in antigenicity of E2 in Semliki Forest virus particles and in infected cells date: 1994 journal: Arch Virol DOI: 10.1007/bf01310027 sha: 52a5f9a9ad01d8a8317599cfc6c09abb3d38082f doc_id: 4923 cord_uid: 0ymcxcj3 Using six monoclonal antibodies to epitopes a-f on the glycoprotein E2 of Semliki Forest virus (SFV) we found antigenic differences between E2 in infected cells and in virus particles, respectively, if glycosylation was impaired by 2-deoxy-D-glucose or inhibited by N-methyl-1-deoxynojirimycin. Furthermore we concluded that a conformational change of E2 takes place on virus budding. Summary. Using six monoclonal antibodies to epitopes a-f on the glycoprotein E2 of Semliki Forest virus (SFV) we found antigenic differences between E2 in infected cells and in virus particles, respectively, if glycosylation was impaired by 2-deoxy-D-glucose or inhibited by N-methyl-l-deoxynojirimycin. Furthermore we concluded that a conformational change of E2 takes place on virus budding. The composition and structure of carbohydrate side chains attached to viral glycoproteins is important for virus maturation. They may influence virus adsorption and penetration, transport of viral glycoproteins [12] , proteolytic cleavage of precursor proteins [1, 7] , association of spike glycoproteins [13] or their antigenicities [5, 11] . With Semliki Forest virus (SFV), a togavirus containing the nucleocapsid protein C and three envelope glycoproteins El, E2 and E3, we previously observed that virus maturation could be inhibited by impairment of glycosylation, e.g. by treatment of infected cells with tunicamycin or 2-deoxy-D-glucose (2-DG) [5] . Using polyclonal antisera, we have shown in addition that antigenicities of glycoproteins with no carbohydrate side chains (after treatment with tunicamycin) or with truncated chains (after treatment with 2-DG) differed from those of virus particles [2, 4, 9-] . Using six monoclonal antibodies (mabs) specific for six epitopes a-f on virion E2 we recently found changes in antigenicity of virion E2, if SFV was propagated in cells in presence of N-methyl-l-deoxynojirimycin (MdN): released, infectious virus particles contained mannose-rich carbohydrate side chains of the composition GI%ManT.8,9(GlcNAc)2; epitope d reacted very well, epitopes c and e reacted less, and epitopes a, b and f only very slightly [6, 10-] . In addition samples of the respective lysates were treated with the rabbit antiserum K, which precipitates all structural components of the virion. Immunocomplexes were removed with protein A-containing Staphylococcus aureus cell walls. After SDS-PAGE (under non-reducing conditions) radioactivity was detected by fluorography. Note that in A additional precipitated bands (c, d, e bands between p62 and E2, and between E1 and E2, respectively) are due to different numbers of attached carbohydrate side chains on E2 or p62, respectively Here we report results obtained in reactions of viral glycoproteins with the same mabs, but in infected cells. Briefly, chicken embryo cells (CEC) were infected with SFV at a moi = 100. The culture medium (MEM with 5% glucose) contained either 5 m M 2-DG, or 2 m M MdN, or no additions. 5.5 h p.i. viral proteins were labeled for 30min with 25gCi 35S-methionine (Amersham Buchler)/2 ml/plastic Petri dish. Lysates were prepared for radioimmunoprecipitation (RIP) as described previously [-6 ] and analyzed with the mabs or with a polyclonal rabbit antiserum K which precipitated all structural SFV proteins. Immune-complexes were subjected to SDS-PAGE according to under non-reducing conditions for separating E1 and E2 [6] . Proteins were detected by fluorography as described previously [6] . After treatment of SFV-infected cells with 2-DG, only epitopes c, d and e were accessible on E2 and on p62 (p62 is the intracellular precursor of E2 and E3) (Fig. 1A) . The same result was obtained after treatment with tunicamycin (not shown). The results indicate that the structure of p62 and of intracellular E2, respectively, differed from that of correct glycosylated proteins. These observations are in agreement with our results obtained previously that sugar-free apoforms of p62 exposed antigenic sites absent in the virion [4] . If the first step of the oligosaccharide processing was blocked by MdN, all epitopes, except f, were accessible (Fig. 1B) . In control infected cells, all epitopes were accessible (not shown). So epitope f only was recognized on E2 with complex carbohydrate side chains, as postulated previously [6] . But it was interesting to observe that the pattern of accessible epitopes after treatment with MdN was not identical for intracellular viral glycoproteins and for E2 of the virion. These results can be explained by a structural and antigenic change occurring during the virus budding process. It indicates that composition of attached carbohydrate side chains on E2 influence virus antigenicity and is important for antibody reactivities. Other authors also reported that, if HIVinfected cells were treated with MdN, the accessibility of virion proteins to mabs was altered as well as bio-activity and conformations [2] . Dissection of Semliki Forest virus glycoproteins delivery from the trans Golgi network to the cell surface in permeabilized BHK cells Effect of a glucosidase inhibitor on the bioactivity and immunoreactivity of human immunodeficiency virus type 1 envelope glycoprotein Inhibition of the multiplication of enveloped RNA-viruses by glucosamine and 2-deoxy-D-glucose Effect of impaired glycosylation on the biosynthesis of Semliki Forest virus glycoproteins Carbohydrate-induced conformational changes of Semliki Forest virus glycoproteins determine antigenicity The significance of carbohydrate trimming for the antigenicity of Semtiki Forest virus glycoprotein E2 Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Inhibition of the multiplication of enveloped viruses by glucose derivates Inhibitors of trimming: New tools in glycoprotein research A carbohydrate side chain on hemagglutinin of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody Intracellular transport of recombinant coronavirus spike proteins: Implications for virus assembly The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation