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 ؋ 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 ؋ 10 ؊7 to 2.3 ؋ 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.A fundamental feature of RNA viruses is the error-prone nature of replication of their genomes, resulting in adaptation and rapid evolution. Various in vitro, in vivo, and ex vivo methods have been used to estimate mutation rates of viral RNA-dependent RNA polymerases. The generally accepted average value of 10 Ϫ4 to 10 Ϫ5 mutations per nucleotide (nt) per round of RNA replication (reviewed in references 6, 7, 23, and 28) results in substitution of 0.1 to 1 nt per each synthesized genomic RNA molecule of ϳ10 kb. Mutation rates vary between 10 Ϫ3 and 10 Ϫ6 in individual studies, depending on the virus and method used, although none of these methods provide an exact measure of the error rate. In vitro methods that utilize purified viral RNA polymerases may overestimate the error rate due to suboptimal in vitro enzymatic conditions and experimental complexity. In ex vivo methods, such as sequencing of phenotypic mutants generated in cell culture and analysis of monoclonal antibody escape mutants or of rates of reversion of phenotypic markers, the number of rounds of RNA replication in infected cells is not precisely known. Methods that involve a reverse transcription-PCR step with cloning of cDNA in bacteria prior to sequencing can be misleading because of the accumulation of additional mutations introduced by reverse transcription-PCR.The Flavivirus genus of the Flaviviridae family consists of about 70 viruses of which 40 have been associated with human illness (reviewed in reference 2). Flaviviruses are small enveloped plus-strand RNA viruses (reviewed in reference 18). The viral particle contains a nucleocapsid composed of viral RNA and capsid protein C, surrounded by a lipid bilayer in which the envelope protein E and membrane protein M are embedded. The genomic RNA is about 11,000 nt long and contains a single long open reading frame (ORF). Translation of the ORF produces a single polyprotein precursor containing viral proteins in the order: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5, where C through E are the structural components of the virion (prM is a pr...

Replication of not-known-vector flaviviruses in mosquito cells is restricted by intracellular host factors rather than by the viral envelope proteins

“…These findings were corroborated by the observation that the MOD/DENV2 chimera replicated efficiently in Vero-B as well as in C6/36 cells (akin to the parent virus DENV2, but unlike MODV) ( (Pletnev et al, 1992;Pletnev & Men, 1998;Charlier et al, 2004). It was suggested that this may relate to as yet poorly understood incompatibilities within the artificially joined chimeric genomes, independent of changes in the virus entry pathways (Pugachev et al, 2004).…”

“…2b) et al, 1992;Pletnev & Men, 1998;Charlier et al, 2004). It was suggested that this may relate to as yet poorly understood incompatibilities within the artificially joined chimeric genomes, independent of changes in the virus entry pathways (Pugachev et al, 2004).From the experiments described above, it appears that the block to productive MODV replication in insect cells (which should finally lead to release of infectious virus progeny) is not at the level of attachment and entry, but rather at a downstream post-entry stage of the virus life cycle. To study this hypothesis, Vero-B and C6/36 cells were transfected with 2 mg MODV RNA isolated from infectious particles (RNeasy Mini kit; Qiagen) using DMRIE-C Reagent (Invitrogen) according to the manufacturer's instructions.…”

“…2b) et al, 1992;Pletnev & Men, 1998;Charlier et al, 2004). It was suggested that this may relate to as yet poorly understood incompatibilities within the artificially joined chimeric genomes, independent of changes in the virus entry pathways (Pugachev et al, 2004).…”

Molecular Basis for Adaptation of a Chimeric Dengue Type‐4/Japanese Encephalitis Virus to Vero Cells

“…NS4B is a membrane-spanning, scaffolding protein which is known to mediate interactions with viral and cellular proteins on both sides of the endoplasmic reticulum membrane (5,15). It may be essential for nucleocapsid-envelope interaction during flavivirus assembly (23). A number of recombinant DEN4 viruses bearing putative Vero cell adaptation mutations were reported, indicating that NS4B protein is a key determinant in the adaptation of DEN4 virus to efficient replication in Vero cells (5).…”

The yellow fever 17D virus as a platform for new live attenuated vaccines

“…37 Despite the high-fidelity of YF17D virus RNA polymerase during viral RNA replication, the introduction of mutations can be detected. 38 Mutations noted at the structural protein level would be related to adaptation of the flavivirus envelope and prM-M protein to the YF17D capsid protein toward virus assembly. In addition, mutations observed elsewhere in the genome could be due to adaptation to growth in cell culture.…”