Jun 10, 1975 - Institute of Technology, Ian Kennedy, Univer- sity of Warwick, and P. Faulkner, Queen's. University, Ontario, Canada. Anti-Sindbis antibody from ...
Vol. 16, No. 4 Printed in U.S.A.
JouRNAL oF VROLOGY, Oct. 1975, p. 1075-1076 Copyright 0 1975 American Society for Microbiology
Sindbis Virus Inhibits Phosphatidylcholine Biosynthesis in BHK-21 Cells DENNIS E. VANCE* AND JULIANA LAM Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, V6T I W5 Canada Received for publication 10 June 1975
Sindbis virus inhibits the incorporation of [methyl-3H ]choline into the phospholipids of BHK-21 cells and also inhibits the activity of the enzyme that catalyzes the final reaction involved in phosphatidylcholine biosynthesis (cytidine diphosphate-choline: 1,2-diacylglycerol cholinephosphotransferase; EC 2.7.8.2). In a previous study we demonstrated that phosphatidylcholine biosynthesis in BHK-21 cells was reduced by infection with Semliki Forest (SF) virus and that this inhibition could be explained by a reduced activity of CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity (2). Prior to our experiments Waite and Pfefferkorn (3) had observed, as a result of Sindbis virus infection, a reduction of [methyl3HFcholine incorporation into phospholipids of chicken embryo fibroblasts but not into the phospholipids of BHK-21 cells. Since Sindbis and SF virus are closely related and only differentiated by immunological techniques, we decided to investigate this apparently different effect on phospholipid metabolism by these two viruses. The results of our studies indicate that both SF virus and Sindbis virus inhibit phosphatidylcholine biosynthesis in BHK-21 cells and both inhibit the activities of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. BHK-21 (C-13) cells were obtained from Flow Laboratories and grown as previously described (2) except that the growth medium was now Dulbecco modified Eagle medium. This medium and all' other culture materials were obtained from GIBCO. Plastic petri dishes (150 by 15 mm) were obtained from Microbiological Associates. SF virus was grown as previously described (2). Stocks of wild-type Sindbis virus were obtained from James H. Strauss, California Institute of Technology, Ian Kennedy, University of Warwick, and P. Faulkner, Queen's University, Ontario, Canada. Anti-Sindbis antibody from rabbit (gamma globulin fraction of pooled rabbit antiserum, 80 mg/ml) was graciously provided by James H. Strauss; antiserum to SF virus and Sindbis virus and preimmune serum were kindly provided by Ian Kennedy.
The procedures for assay of the cholinephosphotransferase activity and for measurement of [methyl-3H ]choline incorporation into phosphatidylcholine were previously described (2). The 1,2-diacylglycerol used in the assay of the cholinephosphotransferase was prepared by phospholipase C digestion of phosphatidylcholine (1). The results (Table 1) demonstrate that 6 h after infection of BHK-21 cells with Sindbis virus the incorporation of [methyl-H Icholine into phosphatidylcholine of the intact cell is reduced by 70%. In addition, the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase from BHK-21 cells is inhibited 45% by infection with Sindbis virus. These data are very similar to those obtained when BHK cells were infected with SF virus (2). Additional results with the cholinephosphotransferase acTAMUE 1. Effect of Sindbis virus on phosphatidylcholine biosynthesis in BHK-21 cellsa [methyl-PH Icholine Coiehs Type of BHK cell
incorporation into photransferase lincororationain lipids of cytoplasmic activityc membranes"
Mock infected Sindbis infected
Sindbis virus/ mockd
1,075,900 295,600 0.27
25.0 14.2 0.57
a Confluent BHK cells were infected with wild-type Sindbis virus (37 PFU/cell), and the incorporation of labeled choline into lipids and the cholinephosphotransferase activity were determined at 6 h postinfection as previously described (2). ° Expressed as disintegrations/minute per milligram of protein. cExpressed as nanomoles of phosphotidylcholine per milligram of protein/15 min. dRatio of values from Sindbis virus- and mockinfected cells.
1075
1076
NOTES
J. VIROL.
TAmZ 2. Effect of antiserum on the infectivity of Sindbis and Semliki Forest virusesa Antiserum prepared Virus against:pepre Virus against:
Semliki Forest Sindbis Semliki Forest Sindbis Semliki Forest Sindbis
Sindbis virus Sindbis virus Semliki Forest virus Semliki Forest virus Preimmune Preimmune
% viable cells
Undiluted'
x10'
x 10-2
x10-'
X10-4
0 100 100 0 0 0
0 75 75 0 0 0
0 0 75 0 0 0
0 0 50 0 0 0
0 0 0 0 0 0
a Sindbis virus (1 ml; 12 PFU) or Semliki Forest virus (1 ml; 7 PFU) was incubated with 0.1 ml of antiserum or diluted antiserum for 30 min at 37 C. BHK cells in Linbro dishes were subsequently infected in duplicate with 0.5 ml of virus suspension, and the percentage of viable cells that remained after 72 h was estimated by observation through a phase-contrast microscope. Dilution of antiserum preincubated with virus.
tivity from Sindbis virus-infected BHK cells were also consistent with those observed with SF virus (2): the rate of the reaction was a linear function with respect to time (20 min) and to protein concentration (0 to 100 ug of microsomal protein); the enzymes from infected and mockinfected cells had similar apparent Km's for 1,2-diacylglycerol; the same degree of inhibition of cholinephosphotransferase by Sindbis virus was observed with multiplicities of infection between 10 and 250 PFU/cell; and the most significant decline in cholinephosphotransferase activity occurred between 4 and 6 h after infection with Sindbis virus. Thus, by the above experiments we have been unable to detect any difference between Sindbis and SF virus inhibition of phosphatidylcholine biosynthesis in BHK cells. Since the above results differ from those reported by Waite and Pfefferkorn (3), it was important to demonstrate that the Sindbis virus preparations were not contaminated by SF virus. The results in Table 2 show that antiserum prepared specifically against Sindbis virus prevented infection of BHK cells by Sindbis virus and did not cross-react with SF virus. Similarly, antiserum to SF virus inhibited SF virus infectivity and did not cross-react with Sindbis virus. Preimmune serum did not alter the infectivity of Sindbis or SF virus. Similar results were obtained with an anti-Sindbis
gamma globulin fraction. From these data we may safely conclude that the Sindbis virus preparations were not contaminated with SF virus. We had hoped that Sindbis virus would not inhibit phosphatidylcholine biosynthesis in BHK cells since this would have demonstrated a unique difference between these two viruses in their effects on the host cell. In addition, we would have been able to devise comparative experiments for elucidation of the mechanism by which lipid synthesis is reduced in SF virus-infected cells. Thus, at the present time the mechanism by which these viruses inhibit phospholipid synthesis and the role, if any, which reduced lipid synthesis plays in SF virus replication remain obscure. This research was supported by a grant from the Medical Research Council of Canada. This work was performed during the tenure of an Established Investigatorship of the American Heart Association (D.E.V.). LITERATURE CITED 1. Hanahan, D. J., and R. Vercamer. 1953. The action of lecithinase D on lecithin. The enzymatic preparation of D-1,2-dipalmitolein and D-1,2-dipalmitin. J. Am. Chem. Soc. 76:1804-1806. 2. Vance, D. E., and D. C. Burke. 1974. Inhibition of 3-sn-phosphatidylcholine biosynthesis in baby-hamster kidney-21 cells infected with Semliki Forest virus. Eur. J. Biochem. 43:327-336. 3. Waite, M. R. F., and E. R. Pfefferkorn. 1970. Phospholipid synthesis in Sindbis virus-infected cells. J. Virol. 6:637-643.
