Cpr7p affects multiple steps during TBSV RNA replication <i>in vitro</i>.

<p>(A) Scheme of the viral RNA recruitment assay based on CFE. The ³²P-labeled TBSV (+)repRNA template was added together with p33/p92 and Cpr7p or its derivatives to CFE prepared from BY4741 yeast. The membrane-association of ³²P-labeled TBSV (+)repRNA template is measured using denaturing PAGE gels (Panel B). Note that ³²P-labeled TBSV (+)repRNA template can inefficiently associate with the membrane even in the absence of p33/p92 (lane 2), likely due to nonspecific binding to an RNA-binding host protein in the membrane. (C) Scheme of the CFE-based TBSV replicase assembly assay. Note that the original template RNA is removed during replicase solubilization/purification. Therefore, an added (−)repRNA is tested for replicase activity for each replicase prep. (D) Complementary RNA synthesis that results in ³²P-labeled TBSV (+)repRNA template is measured using denaturing PAGE gels. (E) Detection of single- and double-stranded RNA products produced in the CFE-based TBSV replication assay. The ratio of ssRNA and dsRNA in the samples are shown. Note that the dsRNA product represents the annealed ³²P-labeled (−)RNA and the (+)RNA, while the ssRNA products represents the newly made ³²P-labeled (+)RNA products.