Date of Award
Doctor of Philosophy
R. W. Beck
W. Stuart Riggsby, A. Brown, R. W. Tennant
Three species of double-strand (ds) RNA, designated RF I, RF II, and RF III in order of decreasing size, were produced by RNase treatment of extracts of chicken embryo cells infected for 6 h with Sindbis virus. These species were identifiable with those reported by Simmons and Strauss (28). At a low level of enzyme (0.001 µg/ml) the major species obtained was RF I. When the enzyme concentration was increased 10-, 100, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concurrent decrease in the proportion of RF I. Only RF I and replicative intermediate RNA (RI) were present in the preparations of isolated ds RNA. Mild RNase treatment of these preparations converted the RI primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. The generation of RF's II and III by RNase resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. These results suggest a RNase-dependent conversion of RF I-like molecules to molecules of RF's II and III. The data support the model for Sindbis virus RNA replication proposed by Simmons and Strauss (28).
RF I was apparently the only naturally occurring ds form of Sindbis virus RNA. However, in the pool of partially purified RF I were cores which could be cleaved by RNase to generate RF's II and III. This finding is consistent with a replication model in which all transcription occurs on a negative strand of the size of the genome of Sindbis virus (28).
A study of the electrophoresis of Sindbis virus single-stranded RNA in polyacrylamide gels suggested an explanation of the disparity in molecular weights (MW's) previously reported for alphavirus RNA's. With HeLa cell 45S, 23S, 28S and 18S RNA's as reference markers, the MW's of Sindbis RNA varied with the percentage of acrylamide in the gels, particularly for the virion RNA. This variation (3.2 x 106 to 45 x 106) may be explained in part by a deviation from linearity in the high MW-region of the calibration curves [log (MW) vs. mobility]. The break occurred in gels of 2.0 and 2.2% acrylamide but not in gels of 2.4 and 2.6% acrylamide. The most accurate determinations of MW were obtained on gels which provided strictly linear calibration curves. It is therefore important to use reference markers which calibrate the gel throughout the required range of MW. The deviation from linearity of the calibration curves for certain gels is discussed.
The rate of change in mobility if Sindbis virion RNA with gel concentration was considerably different from the changes in rates of Sindbis messenger RNA and HeLa cell RNA's used for reference. Certain theoretical considerations suggested the possibility that the virion RNA may have a secondary structure very different from those of the other RNA's.
Martin, Jonathan D., "Intrerconversion of Replicative Forms of Sindbis Virus RNA and the Electrophoresis of Viral RNA. " PhD diss., University of Tennessee, 1976.