VIRAL GLYCOPROTEINS AT DIFFERENT STAGES OF INTRACELLULAR-TRANSPORT CAN BE DISTINGUISHED USING MONOCLONAL-ANTIBODIES

Abstract

Mouse monoclonal antibodies were raised against Semliki Forest virus (SFV) and all were found to recognize the E2 spike glycoprotein. The hybridomas were screened by immunofluorescence microscopy of infected [hamster kidney BHK-21] cells to look for antibodies recognizing E2 at different stages of intracellular transport and 2 (9AB4 and 9AD6) were chosen for further study. Immunocytochemical studies at the light microscopic and EM levels were used to show that 9AB4 recognized E2 at all stages of its transport from the rough endoplasmic reticulum (ER), where it was synthesized, to the cell surface via the smooth ER and Golgi complex. 9AD6 only recognized E2 once it had left the ER and entered the stacks of flattened Golgi cisternae. Binding of 9AD6 to E2 did not appear to be the result of the changes in oligosaccharide structure that occur in the Golgi complex and the precise modification detected by 9AD6 remains to be elucidated. A later modification, the cleavage of precursor E2 (p62) to give mature E2, was used to show that 9AD6 bound the latter more tightly than the former; 9AB4 bound both with equal affinity. These results show that monoclonal antibodies might be useful when searching for new modifications to proteins undergoing intracellular transport.