Figure 2.

Codon usage and its relationship to the rate of translation and replication capacities of two variants derived from the HM175 strain of hepatitis A virus (HAV) (HM175-43c: L0 parental type; HM175-HP: HP fast-growing type) that differ in their level of codon optimization. (A) Relative codon deoptimization index (RCDI) of the L0 and HP strains with respect to the human codon usage. RCDI values shown correspond to overlapping 100 codon segments of the capsid-coding RNA with 15 codon-sliding windows. The higher the RCDI value, the higher the deviation of viral codon usage from host cell codon usage. The HP strain shows a remarkable decrease in RCDI values in a region extending 50% of the VP1 length, compared to the L0 strain, which denotes more optimized codon usage relative to the cell. (B) Rate of translation elongation of the VP1 fragment showing differences in the RCDI between L0 and HP strains. The elongation rate is measured as the relative FLuc/RLuc activity, using a bicistronic vector in which Renilla luciferase (RLuc) translation is cap dependent and Firefly luciferase (FLuc) translation is HAV internal ribosome entry site (IRES) dependent. The VP1 fragment is cloned under control of the IRES just upstream of and in frame with FLuc. Translation elongation was assayed using two bicistronic vectors representing the parental-type IRES (L0-type IRES: two first points of the kinetics) and the mutated-active-type internal ribosome entry site (IRES) (HP-type IRES: two last points of the kinetics), under conditions of no shutoff (–) or shutoff (+). Results are expressed as fold change of the elongation rate relative to the L0 population with the parental-type IRES and in the absence of shutoff. (C) Box plots of the virus yields obtained in FRhK-4 cells. (D) Box plots of virus yields obtained in HuH7-AI cells. In both C and D, the multiplicity of infection was 1. Dotted lines represent mean titer.