Figure 4.

 The cloverleaf structure formed at the 5′ end of the viral genome controls viral translation. (A) Schematic representation of the ribonucleoprotein complex formed around the cloverleaf RNA. The predicted cloverleaf structure is composed of stem–loop B (nucleotides 10–34), stem–loop C (nucleotides 35–45), and stem–loop D (nucleotides 51–78). Viral factor 3CD and cellular protein PCBP are shown interacting with their specific target sequences. The locations of the mutations introduced into the cloverleaf structure of the Polio–Luc RNAs are indicated by arrows: LB.14 (nucleotides 23–26, CCCA, were deleted in loop B); SB.212 (nucleotides 14–16, GGG, and nucleotides 28–30, CCC, were replaced with AAA and UUU, respectively, which maintain the stem B structure); and LD.73 (nucleotides GUAC were inserted in position 70 of loop D). (B) Luciferase activity produced by Polio–Luc constructs carrying wild-type or mutated cloverleaf structures. In vitro-transcribed Polio–Luc RNAs were either transfected into HeLa cells (top) or microinjected into Xenopus oocytes (bottom). The RNAs are indicated as WT (wild-type), ΔCL (cloverleaf-deleted), LB.14 (loop B muted), SB.212 (stem B mutated), and LD.73 (loop D mutant). Luciferase activity was measured in HeLa cell extracts 2 hr after electroporation and in oocyte extracts 10 hr after injection, and expressed in arbitrary units (AU). (C) Microinjection of decoy cloverleaf RNAs into Xenopus oocytes interferes with poliovirus translation. Wild-type Polio-Luc RNA was coinjected with buffer (−), 30 ng of wild-type cloverleaf (WT), or 30 ng of mutant cloverleaf decoys (LB, nucleotides C23 to A26 deleted, or LD, nucleotides GUAC inserted in position 70). Luciferase activity was determined in oocyte extracts 10 hr after injection.