Figure 2.

Modifications Were Introduced into 3D^pol to Increase Replication Fidelity and Reduce Recombination Rate

(A) To identify high-fidelity variants, Sabin2 virus was grown in HeLa S3 cells at m.o.i. of 0.1 with and without ribavirin under 33°C for 10 h. After eight passages, virus populations were analyzed by CirSeq (Acevedo et al., 2014) to identify fidelity determinants.

(B) Distribution of the 11 mutations revealed by CirSeq analysis as potential fidelity determinants on 3D^pol structure.

(C) To test for replication fidelity, we used a ribavirin-sensitivity test to determine the inhibitory concentration 50 (IC₅₀). Among the 11 fidelity candidates, HiFi3 (D53N) increased resistance the most to the mutagen ribavirin. The mean values calculated were significantly higher for Sabin-D53N than for Sabin2, with 95% confidence intervals that do not overlap.

(D) To identify recombination-rate determinants, the eGFP gene was inserted into the Sabin2 viral genome between P1 and P2 regions to generate Sabin2-eGFP virus. Serial passages of the Sabin2-eGFP virus often lead to eGFP loss because of homologous recombination, but variants with reduced recombination rates can be isolated by selecting eGFP-positive clones.

(E) After five passages in HeLa S3 cells, five potential recombination determinants were identified as shown on the 3D^pol structure.

(F) Experimental design of the “recombination validation assay.” Viral RNAs with either mutated CRE or truncated 3D RNA polymerase do not produce viable viral progeny when transfected into L929 cells individually. Viable virus may be produced by recombination between the two truncated RNAs.

(G) Titers of viral progeny from the “recombination validation assay.” Candidate mutations K38R, P243S, and L445M reduced recombination to 29.0%, 75.8%, and 38.7% to the parental strain Sabin2 (WT), respectively. Asterisk indicates significant difference as compared with WT; p values are 0.0322, 0.2277, and 0.0313 for K38R, P243S, and L445M, respectively. Data are shown as mean with SEM.

(H) Effect of HiFi (D53N) and Rec1 (K38R) mutations on Sabin2 replication was initially determined using a luciferase replicon replication assay. Data are shown as mean with SEM. ^∗p = 0.0187.

(I) Effect of HiFi (D53N) and Rec1 (K38R) mutations on Sabin2 virulence was evaluated by PD₅₀ of each mutant virus determined in PVRTg21 mice.