Membrane phospholipid asymmetry in Semliki Forest virus grown in BHK cells

The distribution of phospholipids across the membrane bilayer of Semliki Forest virus grown in BHK cells has been examined by treating the virus with bee venom phospholipase A₂ and sphingomyelinase C from Staphylococcus aureus. From the amounts of different phospholipids which are degraded rapidly (half-time about 1 min for phospholipase A₂) we calculate that in virus isolated 16 h after infection about 95% of sphingomyelin, 55% of phosphatidylcholine, 20% of phosphatidylethanolamine and less then 5% of phosphatidylserine is present on the outer leaflet of the virus envelope. Less than 5% of the virus was permeable to macromolecules before or after treatment with phospholipases as judged by accessibility of the genome to external ribonuclease. A much slower (half-time about 1 h) breakdown by phospholipase A₂ of originally inaccessible phosphatidylcholine and phosphatidylethanolamine appeared to be due to an enzyme-induced loss of lipid asymmetry since the original asymmetric distribution of phospholipids was maintained for several hours when the virus alone was incubated at 37°C. However, virus incubated for 20 h at 37°C showed a marked loss of phosphatidylethanolamine and phosphatidylserine asymmetry and a greater susceptibility to lysis by longer treatment with phospholipase A₂.