The effect of Semliki Forest virus infection on phosphatidylcholine biosynthesis in baby hamster kidney-21 cells

Frederick William Whitehead
Semliki Forest (SF) virus caused an inhibition of 77% in incorporation of [³H] choline into phosphatidylcholine (PC) of Baby Hamster Kidney -21 (BHK) cells, at 6½-7½ hours post infection (p.i.). Choline uptake, enzyme activities, and pool sizes were measured to clarify the mechanism of inhibition and to understand the regulation of PC synthesis. Choline uptake has a K[sub m] of 17 μM and V[sub max] of 381 pmoles min⁻¹ mg cell protein⁻¹ in mock-infected (control) cells. Uptake is inhibited in
more » ... is inhibited in infected cells, although such inhibition only partly accounts for incorporation inhibition. Maximal velocities of the enzymes of de novo PC synthesis in nmoles min⁻¹ g cells⁻¹, from control cells, were: choline kinase - 7.3; cytosolic phosphocholine cytidylyltransferase (cytosolic CT) -17.3; microsomal CT - 14.6; and cholinephos-photransferase (CPT) - 47.6. In infected cells, the respective activities were less: 5.2, 12.1, 4.2, and 19.8, at 7 hours p.i.. Choline, phosphocholine, and CDP-choline were separated by ion exchange and charcoal chromatography. Phosphocholine and CDP-choline were hydrolyzed to choline, which was measured enzymically. Diglyceride was hydrolyzed to glycerol, which was measured enzymically. CTP was measured by a new enzymic technique which uses rat liver CT. ATP was measured by its absorbance after high pressure liquid chromatography. PC was measured by lipid phosphorus analysis. Pool sizes in nmoles/g cells, from control (and infected) cells were: choline - 146 (68); phosphocholine - 34 (120); CDP-choline - 6.1 (15.7); diglyceride - 47 (43); CTP - 149 (79); and ATP - 1800 (1080), all at 6½-7½ hours p.i.. Increases in phosphocholine and CDP-choline, and decreases in CTP and ATP, were all significant (p<0.05). The pool size of PC, in μmoles/g cells, was 3.4 in control cells, and similarly, 3.0 in infected cells, at 7-7½ hours p.i.. The fatty acid composition of both PC and diglyceride was very similar in control compared to infected cells. In BHK cells which were labelled [...]
doi:10.14288/1.0094808 fatcat:t43f6zb5v5audn7wxdizavrubq