Genome 3'-end repair in dengue virus type 2

Abstract

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3'-end repair in dengue virus-2 in mammalian cells, a series of 3'-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7-10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3' end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3'-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3' stem-loop (3' SL) structure was abrogated by mutations, viruses eventually restored the 3' SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3'-end sequences based on the best-fit RNA structure that can support viral replication.

FIGURE 1.

Immunofluorescence assay (IFA) to monitor virus replication. In vitro transcribed RNAs were used to transfect LLC-MK2 cells by electroporation. Cells were stained with a monoclonal antibody specific for DENV2 NS1 protein.

FIGURE 2.

Plaque assays of recovered viruses from parental (WT+G) and 3′-deletion mutants (Δ4+GC and Δ10+GGC) input RNAs. Viruses were collected from supernatants on the indicated days and plaque titers were determined. The plaque assays were repeated three times and the plaque titers (pfu/mL) of a representative experiment are given. The average plaque sizes (mm) at indicated days are as follows. WT: day 20: 8.0 × 10³, 2.64 ± 1.06 mm. Δ4+GC RNA: day 13: 1.2 × 10⁴, 1.25 ± 0.44 mm; day 20: 1.1 × 10⁴, 1.86 ± 0.57 mm; day 27: 7.5 × 10⁴, 2.41 ± 1.18 mm; day 48: 1.5 × 10⁴, 2.67 ± 0.81 mm. Δ10+GGC: day 48: 4.0 × 10³, 1.52 ± 0.51 mm; day 62: 8.5 × 10³, 2.31 ± 0.83 mm.

FIGURE 3.

In vitro template activity of sgRNAs containing WT or 3′-end deletions. sgRNAs containing WT or 3′-end deletions were analyzed for template activities in vitro using purified NS5. (A) sgRNAs containing Δ4+GC (lane 1), Δ8+GGC (lane 2), Δ10+GGC (lane 3), Δ14+GGC (lane 4), Δ17+GC (lane 5), and Δ22+GC (lane 6) were used as templates for negative-strand synthesis. (Lane 7) No RNA control. The products of the reaction were separated by electrophoresis and visualized by autoradiography. (1×) De novo product, (2×) double-stranded hairpin product. Ethidium bromide gel shows approximately equal amounts of sgRNAs used for RdRp assays. Experiments were repeated at least three times and the results of a representative experiment are shown. (B) Densitometric scan of gels were performed, and the sum of 1× and 2× RNAs was compared with WT. Error bars are based on three independent experiments. (C) Densitometric scan of 2× product only, compared with that of WT. (D) Densitometric scan of 1× RNA compared with the WT. (E) In vitro template activity of sgRNAs bearing adaptive mutations identified in recovered genomes. sgRNAs bearing 3′-end sequences, WT and 1–8, were used as templates in the in vitro assays. The RNA products were detected by autoradiography.

FIGURE 4.

Analysis of secondary structures by Mfold. Structures I (A) and II (B) are similar to those described by Alvarez et al 2005. (C) Structure I representation of WT and consensus viral genomes recovered from Δ4+GC, Δ7+GGC, Δ8+GGC, Δ10+GGC viral RNAs, respectively, from left to right. (Shaded nucleotides) Substitution mutations, (boxes) recovered nucleotides. (D) Input RNA transcripts of Δ6, Δ6A, Δ6B, and Δ6C (see Table 3) are shown at left. The structure I representation of recovered genomes is shown on the right. Δ6A-derived viral genomes were classified as three types, based on the sequence alignments (see Table 3). Δ6B-derived genomes have a single sequence type, although they have variable lengths. Δ6C-derived genomes are classified as three sequence types. The secondary structures were drawn using the Mfold program (see Supplemental Table 3).