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C6/36 (Aedes aegypti) Cells

WT HP3.4 HP11.9

WT HP3.4 HP11.9

FIG. 2. Regulation of DENV replication by a coding region structural element. (A) The predicted secondary structure of the first 150 nucleotides of DENV-2 showing the cHP. The RNA secondary structure of the first 150 nucleotides of DENV-2 was predicted by mFold; the start codon and the cHP are indicated by boxes. (B) Schematic of infectious clone (IC) variants utilized to study the role of the cHP and of the nucleotides that make up the DENV-2 initiation context. The pD2/IC DENV-2 IC was a gift of R. Kinney (Centers for Disease Control and Prevention, Fort Collins, CO). (C) Viral titres from IC-transfected Hep3B cells. In vitro-transcribed RNAs were transfected into Hep3B cell monolayers, and viral replication was assessed after 72 h by plaque assay. Titres were normalized to transfection efficiency as determined by qRT-PCR at 2 h post-transfection. Viral titres are expressed as plaque-forming units per ml (PFU/ml) from IC-transfected Hep3B cells. One log reflects the limit of detection of a standard plaque assay. Error bars indicate SD; data derived from four experiments. (D) Viral titres from IC-transfected C6/36 cells. Methods and graph as in C; data are derived from four experiments.

Congruent with the conservation of the cHP among flaviviruses, this cis element is necessary for replication of DENV-2 in Hep3B and C6/36 cells. To test the requirement for the DENV-2 cHP in the viral lifecycle, mutations were made in the infectious clone of DENV-2 prototype Thai strain 16681 (pD2/IC) that were predicted to disrupt the cHP structure (Fig. 2B). Viral RNA was in vitro-transcribed and transfected into Hep3B and C6/36 cells. Viral replication was assessed after 72 h by plaque assay and normalized to transfection efficiency as measured by quantitative RT-PCR of infectious clone RNA at 2 h post-transfection. In Hep3B and C6/36 cells, disruption of the cHP (pD2/IC-HP3.4) reduced viral replication by at least 2.5 logs and nearly 4 logs, respectively, to levels undetectable by plaque assay (Fig. 2C and 2D). Restoration of the hairpin structure (pD2/IC-HP11.9) restored viral replication to levels similar to wild-type (Fig. 2C and 2D). Similarly, residues at the —3 and +4 positions relative to the start codon, the first start codon and the second in-frame start codon are all required for efficient viral replication in both cell types (data not shown). These data demonstrate that coding region RNA secondary structure plays a regulatory role in translation and replication of DENV and perhaps other flaviviruses.

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