Figure 12.

VP5 facilitates RT initiation via a CPV panhandle. (A) Schematic illustrations of the predicted panhandle structure formed by 36 bases from the 5′-end and 30 bases from the 3′-end of HaCPV-5 RNA segment 8 (upper panel) and the primer complementary to the 3′-end of the panhandle (lower panel). (B) The RT reactions were conducted using the RNA panhandle structure and the DNA primer in the presence of M-MLV reverse transcriptase for 30 min at 25°C in the absence (lanes 1 and 4) or presence of MBP-VP5 (lane 2) or MBP alone (lane 3). For lane 1, the panhandle and primer were prehybridized via a thermal annealing treatment at 68°C before the reaction. (C) The RT reactions were conducted using the indicated amounts of RNA panhandles in the presence of indicated amounts of MBP-VP5 for 30 min at 25°C. The samples were analyzed on 6% urea PAGE, followed by northern blotting.