FIG. 4.

Functional in vivo and in vitro compatibility of SLAs from different DENV serotypes. (A) Enzymatic probing of the first 70 nucleotides of DENV1 and DENV2. The RNAs were subjected to RNase V₁ (V) or RNase A (A) digestion, and the cleaved RNAs were analyzed by primer extension. Differences within the S3 and SSL regions of SLAs from DENV1 and DENV2 are indicated. (B) In vitro activity of viral RdRp for WT and mutated RNA templates with 160 nucleotides. (Top) Sequences of SLAs of the RNAs used as templates for RNA synthesis. (Bottom) Viral polymerase activity, expressed in fmol [α-³²P]GMP incorporated into acid-insoluble RNA per minute and per μg of protein, for each RNA template. The reaction was carried out as described in Materials and Methods. Error bars indicate the standard deviations of results from three experiments. (C) Expression of DENV proteins in BHK cells transfected with RNA of WT DENV2, a chimeric DENV carrying the SLA from DENV1 (DENV1/DENV2), and mutants with deletions of the SSL or S3 (Mut ΔSSL or Mut ΔS3, respectively) was determined. Viral replication was monitored by IF for up to 12 days posttransfection.