Infectivity of Artificially Nicked Viroid Molecules

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Abstract
Circular molecules of potato spindle tuber viroid (PSTV) were subjected to limited digestion by various methods to produce nicked molecules. The resulting linear molecules were separated electrophoretically from circular ones, and assayed for infectivity. The linear molecules produced by treatment with RNase CI or RNase U2 were as infective as circular molecules. These linear molecules had 5′-OH and 3′-phosphate ends; almost all 3′ termini probably were in the form of 2′,3′-cyclic phosphate. However, the linear molecules produced by treatment with RNase T1 were approximately 10-fold less infective than circular molecules. The 2′,3′-cyclic phosphate at the 3′ end of these linear molecules had presumably been partially converted to 3′-phosphate. Linear molecules produced by a Mg2+-catalysed nicking reaction had a mixture of 2′- and 3′-terminal phosphates at their 3′ end and were approximately 100-fold less infective than circular ones. The infectivity of linear molecules produced by nuclease S1 digestion which lacked a 3′-phosphate was 100- to 1000-fold less than that of circular ones. These results indicate that the 3′-terminal phosphate of linear PSTV molecules is required for infectivity, and for maximum infectivity is required to be in the form of 2′,3′-cyclic phosphate.