High fidelity of yellow fever virus RNA polymerase

Abstract

Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 x 10(-4) per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 x 10(-7) to 2.3 x 10(-7). Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

FIG. 1.

Passages of the cloned and uncloned ChimeriVax-DEN2₂₀₀₀ viruses. P1 virus was obtained by electroporation of Vero cells with in vitro RNA transcripts synthesized using SP6 RNA polymerase. Uncloned P2 virus was generated by one additional passage. Cloned PMS candidates (clones A and B) were produced by three consecutive plaque purifications starting from P1 virus followed by two additional passages. Genetic stabilities of these cloned and uncloned variants were examined in additional downstream virus passages. ChimeriVax-DEN2₂₀₀₁, -DEN3, -DEN4, and -DEN1 viruses were passaged similarly, except that plaque purification passages (P3 to P5) were initiated from the uncloned P2 viruses.

FIG. 2.

Clustering of beneficial amino acid changes in the four ChimeriVax-DEN chimeras that accumulated in all cloned and uncloned viruses during genetic stability passages. The locations of mutations in the polyprotein and genomic RNA in each of the chimeras were plotted according to their positions in the genome.