Fig 3.

Delimitation of the DHAV 5′ UTR sequences required for IRES activity in BHK cells. The cDNA fragments indicated were generated by using the PCR as described in Materials and Methods and inserted into the XhoI and NcoI-digested vector pRL-NO. Dicistronic luciferase plasmids with 5′-terminal (A), internal (B), and 3′-terminal (C) deletions made in the DHAV 5′ UTR and inclusion of 30 nt of coding sequence downstream of the DHAV 5′ UTR were used to transfect vTF7-3-infected BHK cells. After 20 h, cell extracts were prepared and analyzed for the expression of Rluc and Fluc. (C and D) The Rluc and Fluc activities were measured and IRES activity was calculated as the mean of three independent experiments. The results were standardized to the values for the Fluc/Rluc ratio directed by the plasmid pRL-DHAV, which was set at 100%. The mean values (plus standard errors of the means [error bars]) are shown.