(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).

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.