FIG. 2.

Translation of VL30m bicistronic RNA in messenger-dependent RRL. (A) Schematic representation of the bicistronic plasmid constructs containing different portions of the VL30 5′ RNA located between the neo and lacZ genes under the control of the T7 promoter (Po T7) for in vitro expression. Numbering is with respect to the genomic RNA cap site (position +1). Po CMV, cytomegalovirus early promoter; KD, kilodaltons. (B) Translation of uncapped (−) and capped (+) bicistronic RNA in the Flexi-RRL system (Promega). After heat denaturation, ³⁵S-labelled proteins were analyzed by SDS–15% PAGE. The positions of neomycin phosphotransferase (28 kDa) and the C-terminally truncated β-Gal protein (46 kDa) are indicated. Lanes 1 to 4, control RNAs containing the EMCV IRES (see Materials and Methods); lanes 7 to 10 RNAs containing different 3′ deletions in the putative VL30m IRES; lanes 5, 6, and 11 to 14, RNAs containing the 5′ region VL30m RNA or 5′ deletions of this sequence.