Step-wise CFE-based TBSV replication assay supports an inhibitory role for Cpr7p in the assembly of the TBSV VRC.
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(A) Scheme of the CFE-based TBSV replicase assembly and replication assays. Purified recombinant p33 and p92pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to CFE prepared from BY4741 yeast strain in step 1. The assay either contained or lacked the purified recombinant Cpr7p, its derivatives (0.4, 0.8 and 1.6 µg), or GST during step 1. Note that the assay was performed in the presence of ATP/GTP to facilitate TBSV VRC assembly, but prevent RNA synthesis in step 1. After step 1, centrifugation was used to collect the membrane fraction of the CFE, and after washing the membranes, step 2 was performed in the presence of ATP/CTP/GTP and 32P-UTP to allow TBSV RNA replication. In the samples presented on the right side of the panel, the recombinant Cpr7p or derivatives were added at the beginning of step 2. (B) Denaturing PAGE analysis of the 32P-labeled TBSV repRNA products obtained in the CFE-based assays when Cpr7p or derivatives were added at the 1st step. See further details in Figure 1. (C) Denaturing PAGE analysis of the 32P-labeled TBSV repRNA products obtained in the CFE-based assays when Cpr7p or derivatives were added at the 2nd step. Three repeats of each experiment were performed.