EUKARYOTIC EXPRESSION OF CLONED cDNA CODING FOR INFLUENZA VIRAL GLYCOPROTEINS USING AN SV40 VECTOR: USE OF RECOMBINANT DNA MUTANTS TO STUDY STRUCTURE-FUNCTION RELATIONSHIPS

Abstract

Publisher Summary Influenza virus has been widely used to study the biosynthesis, sorting, distribution, and orientation of membrane proteins. Influenza has three membrane associated proteins; hemagglutinin (HA), neuraminidase (NA), and matrix protein (M). HA and NA are integral membrane proteins located on the outer envelope, whereas M protein is associated with the inner lining of the viral membrane. As influenza contains these two types of integral membrane proteins, it is an ideal candidate for the study of the mechanism by which integral membrane proteins are transported from their site of synthesis, via the rough endoplasmic reticulum and golgi apparatus, to their final destination in eukaryotic cells. Co-translational and post-translational modifications along with specific structural features of a protein are presumably responsible for guiding the protein through the elaborate cellular machineries to its final destination. The conserved residues may, however, play an important role in sorting directional transport or interactions with other viral proteins such as M in the viral membrane. This chapter describes methods used to express HA and NA in CV1P cells by using SV40 as a vector. The cDNA is cloned into the late region of SV40 between Hpa II and Bam HI, replacing the late SV40 genes. CV1P cells are transfected with the recombinant virus and SVsal.32—a helper virus defective in the early genes.