Identification of Single- and Double-Stranded RNAs Associated with Maize Stripe Virus

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Abstract
Purified nucleoprotein from maize stripe virus (MStpV)-infected plants formed a single band with a density of 1.27 g/cc[cm2] in cesium sulfate equilibrium density gradients, but sedimented in rate-zonal sucrose density gradients as 4 major nucleoprotein components with sedimentation coefficients between .apprx. 70 and 187S. The purified nucleoproteins from both the cesium sulfate and sucrose gradients were infectious. A complex mixture of both single- and double-stranded (ss- and ds-, respectively) RNA was identified in all purified nucleoprotein preparations of MStpV when RNA were extracted with SDS and phenol and analyzed by nondenaturing gel electrophoresis. Electrophoresis of denatured RNA showed 5 RNA of .apprx. 3.01, 1.18, 0.81, 0.78 and 0.52 .times. 106 relative mass (MW). The ds-RNA species detected in purified nucleoprotein preparations coelectrophoresed with ds-RNA extracted from MStpV-infected tissues, except that one small ds-RNA (.apprx. 0.66 .times. 1-6 MW) was found in tissue extracts and not in nucleoprotein samples. The nucleoprotein ds-RNA were estimated to be .apprx. 4.9, 2.58, 1.73, 1.67, and 0.87 .times. 106 MW under nondenaturing conditions. Denatured ss- and ds-RNA separated by 2 M LiCl fractionation had identical mobilities, suggesting that the ds-RNA are complementary to the respective ss-RNA. The sizes of the ss- and ds-RNA in individual nucleoprotein components were proportional to the sedimentation coefficients. The nucleoprotein 0.52 .times. 106 ss- and 0.87 .times. 106 ds-RNA were isolated from the slowest-sedimenting component (70S), while the 3.01 .times. 106 ss- and 4.9 .times. 106 ds-RNA were isolated from the fastest-sedimenting component. Nondenaturing electrophoresis of RNA isolated and electrophoresed immediately from purified individual nucleoprotein components showed mostly ss-RNA, whereas SDS [sodium dodecyl sulfate] and phenol extraction yielded both ss- and ds-RNA. The possibility that only ss-RNA are encapsidated during virion assembly, and that ds-RNA arise during RNA extraction is discussed.

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