Effect of altered codon pair bias on translation. (A) Structure of a dicistronic reporter (5). The first cistron uses the hepatitis C virus (HCV) IRES to initiate translation of Renilla luciferase (R-Luc). This first cistron provides an internal control to normalize the amount of input RNA. The second cistron uses the poliovirus IRES to initiate translation of Firefly luciferase (F-Luc). The region labeled P1 was replaced by the recoded, synthetic P1 regions of the indicated viruses. (B) Each dicistronic RNA was transfected, in the presence of 2 mM guanidine hydrochloride [to block replication of the transfected genome (7)], into HeLa R19 cells, and after 6 hours the R-Luc and F-Luc were measured. The F-Luc/R-Luc values are expressed relative to the wild type, which was set to 100%. The graph displays the average (±SD) of three independent experiments.

(A) The poliovirus genome. Shown is the viral RNA with its covalently linked 5′ viral protein VPg, the 5′ nontranslated region [consisting of cloverleaf and internal ribosomal entry site (IRES)]; the long open reading frame (open box) encoding the poly-protein that is cleaved into P1 (capsid precursor), P2, and P3 (precursors to nonstructural proteins); and the 3′ nontranslated region terminated with poly(A) (7). The polypeptide precursors P1, P2, and P3 are processed into functional proteins by virus-encoded proteinases (7). Cre is a stem-loop structure functioning as a cis-acting replication element. (B) Calculated codon pair bias (CPB) score for all 14,795 annotated human genes. Each dot represents the calculated CPB score of a gene plotted against its amino acid length. Underrepresented codon pairs yield negative scores. Various poliovirus constructs are represented by symbols; PV(M)-wt is the wild-type poliovirus (CPB = −0.02). (C) The structures of the various chimeric, partly synthetic poliovirus constructs, and their viability on cultured cells. Nucleotide positions are shown. (D) A one-step growth curve with respect to PFUs. An MOI of 2 was used to infect a monolayer of HeLa R19 cells. Symbols: open squares, PV(M)-wt; solid circles, PV-Max; open diamonds, PV-Min755–1513; asterisks, PV-Min1513–2470; solid diamonds, PV-MinXY; open triangles, PV-MinZ. (E) As (D), but with results graphed with respect to viral particles instead of PFUs. (F) Plaque phenotypes of viruses after 72 hours of incubation, stained with crystal violet (plate diameter, 35 mm).