Phospholipid asymmetry in Semliki Forest virus grown on baby hamster kidney (BHK-21) cells

Abstract

The distribution of the different phospholipid classes over the two leaflets of the membrane of Semliki Forest virus, grown on baby hamster kidney cells (BHK-21), was studied. To localize the phospholipids we have used a phosphatidylcholine-specific exchange protein, a nonspecific exchange protein, phospholipases A2 and C, sphingomyelinase, and the amino group labeling reagent trinitrobenzenesulfonate. When low concentrations of exchange proteins and phospholipases were used, only those phospholipids present in lysed or otherwise defective virions were detected. Up to 10% of the phospholipids are present in such particles, and this amount correlated well with the amount of ribonuclease-degradable RNA present in each virus preparation. In the actual localization experiments, carried out with higher concentrations of exchange proteins and phospholipases as well as with trinitrobenzenesulfonate, the various independent techniques yielded identical results. The outer membrane layer of the virus contains 52% of the phosphatidylcholine, 22% of the phosphatidylethanolamine, and 33% of the sphingomyelin. Phosphatidylserine could not be localized. Altogether 30% of the total phospholipids could be assigned to the outer layer and 50% to the inner membrane leaflet whereas 20% could not be localized. Calculations, based on the size of the Semliki Forest virus and the number of phospholipid molecules per virion, indicate it to be unlikely that the phospholipids which could not be localized are present in the inner bilayer leaflet. The present data on phospholipid composition and distribution have been compared with previous results on the lipid distribution in plasma membrane derived structures. It is concluded that an asymmetric distribution of phospholipids in the plasma membrane of various mammalian cells is apparent.