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KMID : 0578319920020010011
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Molecules and Cells
1992 Volume.2 No. 1 p.11 ~ p.16
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In Vitro Replication Assays for identification of Cis-acting Sites in S733 Double Stranded RNA Virus of yeast
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Lee Myunghee
kang Hyen-Sam
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Abstract
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Defective interfering viral particles of yeast, designated S, contains fragments of M1 dsRNA which codes for excreted polypeptide toxic to yeasts without M1 particles, and displaces M1 when they arise. Previous sequence analysis of 5733 revealed that it retains a small 5´ end portion of the plus strand RNA, and a large 3 end portion which are likely to be recognized by viral and host polypeptides necessary for viral propagation. We employed reconstituted in vitro replication assay to identify cis-acting sites necessary for replication in the 3´ end region. In the assays, empty Ll viral particles converted externally added single stranded S733 RNA template synthesized by SP6 RNA polymerase run-off transcription into double stranded RNA. (LI dsRNA codes for capsid proteins and RNA dependent RNA polymerase for L1, M1 and S particles.) The radio-labeled RNA synthesized in the replication reaction was shown to be double stranded by its complete digestion with pancreatic RNase in the absence of 0.6 M NaCl. It is also shown that replicase activity of empty viral particles in vitro presents the same template specificity as in vivo. Template RNAs with different 3´ ends were prepared by transcription of the templates linearized with different restriction enzymes. Then the effect of the changes at the 3´ end of plus strand on replication activity was followed. The result showed that at least the last four bases ATCA and the secondary structure at the 3´ end are important for replication in vitro by empty particles, specifically by RNA dependent RNA polymerase.
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