Identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virus-infected cells

Virosomes are cytoplasmic sites of replication of vaccinia virus DNA and we re prepared from virus-infected HeLa cells. The early virosomal proteins we re S-35-labelled and SDS polyacrylamide gel electrophoresis revealed the pr esence of three major early 35S-labelled proteins of 34, 24 and 45 kDa. The masses of molecules present in the 34 and 24 kDa proteins were measured by the convenient and sensitive MALDI TOE: mass spectroscopy technique. Ident ification of the three virosomal proteins was carried out by MALDI mass spe ctroscopy of corresponding tryptic digests. For each protein at least 13 me asured masses matched, within less than 0.1 Da, calculated tryptic peptides of the vaccinia virus proteins H5R (34 kDa), E3L (24 kDa) and E5R ( 45 kDa ). In addition, virosomes contained several structural proteins from the in fecting virus and a 45 kDa keratin-related protein. This work demonstrates directly that the abundant early vaccinia virus proteins H5R, E3L and E5R a re associated with the virosomes. (C) 1999 Elsevier Science B.V. All rights reserved.