The effect of Semliki Forest virus infection on phosphatidylcholine biosynthesis in baby hamster kidney-21 cells
Creators: name: Whitehead, Frederick William
Publisher: name: University of British Columbia
Year: 2010

description: 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 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&lt;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 with Q3H~J choline, the specific radioactivity and half-life of choline, compared to; phosphocholine, suggested that a large pool of choline exists, which is not active in PC synthesis. In a pulse-chase experiment with [³H] choline, the fraction of the [³H] phosphocholine pool which turned over per unit of time (k) was smaller in infected cells, yet the pool size of phosphocholine was greater, compared to control cells. Consequently, the turnover rate of phosphocholine (k x pool size) in nmoles min⁻¹ g cells⁻¹, was 1.51 in infected cells, and a little less, 1.05, in control cells. Thus, the turnover of phosphocholine (or rate of PC synthesis) was not inhibited by SF virus infection. In BHK cells, the three enzymes of de novo synthesis of PC likely catalyze near-equilibrium (not rate-limiting) reactions because: 1. V[sub max] of each enzyme is much greater than the pathway flux. 2. Pools of the enzyme substrates of choline kinase and CT do not appear to be great enough to saturate the enzymes. 3. All three enzymes are reduced in activity by virus infection, yet the pathway flux is not reduced. If CT catalyzes a near-equilibrium reaction, then a smaller CTP pool would lessen the flux over the CT step. Phosphocoline accumulation would restore this flux. Similarly, CDP-choline accumulation may restore the flux over the CPT step (which was possibly lessened by an increase in amount of the product, CMP). If indeed, the enzymes of de novo synthesis of PC catalyze near-equilibrium reactions, then a change in any substrate or product will change the pathway flux, unless a response in another substrate or product balances the initial change.