RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome

Abstract

Double-stranded RNA (dsRNA) initiates cellular posttranscriptional responses that are collectively called RNA silencing in a number of different organisms, including plants, nematodes, and fruit flies. In plants, RNA silencing has been associated with protection from virus infection. In this study, we demonstrate that dsRNA-mediated interference also can act as a viral defense mechanism in mosquito cells. C6/36 (Aedes albopictus) cells were stably transformed with a plasmid designed to transcribe an inverted-repeat RNA (irRNA) derived from the genome of dengue virus type 2 (DEN-2) capable of forming dsRNA. Clonal cell lines were selected with an antibiotic resistance marker and challenged with DEN-2. The cell lines were classified as either susceptible or resistant to virus replication, based on the percentage of cells expressing DEN-2 envelope (E) antigen 7 days after challenge. Eight out of 18 (44%) cell lines designed to express irRNA were resistant to DEN-2 challenge, with more than 95% of the cells showing no DEN-2 antigen accumulation. One of the DEN-2-resistant cell lines, FB 9.1, was further characterized. DEN-2 genome RNA failed to accumulate in FB 9.1 cells after challenge. Northern blot hybridization detected transcripts containing transgene sequences of both sense and antisense polarity, suggesting that DEN-2-specific dsRNA was present in the cells. In addition, a class of small RNAs 21 to 25 nucleotides in length was detected that specifically hybridized to labeled sense or antisense DEN-2 RNA derived from the target region of the genome. These observations were consistent with RNA silencing as the mechanism of resistance to DEN-2 in transformed mosquito cells.

FIG. 1.

Maps of pIE1-null plasmid and derivatives used to confer DEN-2 resistance in transformed C6/36 mosquito cells. (A) Map of the DEN-2 genome showing the locations of the prM sequences used in this study. Nucleotide position numbers for the Mnp and prM290 sequences are given. (B and C) The pIE1-null plasmid was used as a backbone for insertion of DEN-2 prM sequences, and the three D2prM-containing constructs are shown. DEN-2 prM sequences were cloned into the XbaI site in sense, antisense, or inverted-repeat orientations. Arrows indicate the hsp70 promoter of the hygromycin resistance gene and the IE-1 promoter driving the prM inserts. The XbaI site downstream from the baculovirus hr5/IE-1 enhancer-promoter, which was used for inserts, and the EcoRI (R1) restriction sites are shown. UTR, untranslated region.

FIG. 2.

Nucleotide sequence of the D2Mnp-D2prM290as insert in plasmid pIE1D2prMir. (A) Sequence of nt 400 to 966 of DEN-2 (New Guinea C) genome RNA (uppercase), followed by a linker region (lowercase), followed by the complementary sequence to DEN-2 RNA nt 448 to 738. Self-complementary regions are underlined. (B) Diagram of the insert transcript, showing formation of a 290-bp double-stranded region. Complementary regions are underlined, and arrows indicate sequence polarity. The 3′ untranslated region (UTR) is a baculovirus genome sequence from the pIE1-3 cloning vector.

FIG. 3.

Resistance of clonal transformed C6/36 cell lines to DEN-2 challenge. Clonal cell lines were selected by resistance to hygromycin and challenged with DEN-2 infection at an MOI of 0.01. The height of bars shows the percentage of cells containing DEN-2 E antigen by IFA 7 days postchallenge. (A) Bars a to j (shaded), cell lines transformed with pIE1-null (H cell lines); bars k to z" (open), cells transformed with pIE1D2prMir (FB cell lines). (B) Bar a, H cell line; bars b to l (stippled), cell lines transformed with pIE1D2prM290s (S cell lines); bars m to z‴ (striped), cells transformed with pIE1D2prMas (As cell lines). Cell lines with <5% E-antigen-containing cells (arrows) were considered DEN-2 resistant.

FIG. 4.

IFA. Untransformed C6/36 cells; null cell line H 9.1; sense cell line S 9.3; antisense cell line As 4.2; and fold-back cell lines FB 2.1, FB 9.1, FB 9.2, and FB 17.2 were stained for DEN-2 E antigen 7 days after challenge with DEN-2. Cells were counterstained with Evans blue so that uninfected cells appear red, while infected cells appear yellow-green.

FIG. 5.

Southern blot analyses of DNA isolated from cell lines FB 9.1, H 9.1, and C6/36. Lane a, DNA from FB 9.1 digested with EcoRI; lanes b and c, DNA from C6/36 cells digested with EcoRI and KpnI, respectively; lane d, DNA from FB 9.1 digested with KpnI. Blots were hybridized with pIE1D2prMir labeled with ³²P. Locations of size markers are indicated at the left.

FIG. 6.

Northern blot analysis of mRNA transcripts produced in FB 9.1 and H 9.1 transformed cell lines. Blots were probed for sense (A and B) or antisense (C and D) D2prM290 sequence. RNA was isolated from H 9.1 (A and C) and FB 9.1 (B and D) cells. Probe was strand-specific, labeled transcript from D2prM290 insert. Ethidium bromide staining of rRNA in each lane is shown below.

FIG. 7.

Slot blot analysis comparing DEN-2 RNA accumulation in FB 9.1 and H 9.1 cells after DEN-2 challenge. Total cellular RNA from each cell line on days 1 to 14 following DEN-2 challenge was blotted and probed for DEN-2 RNA, with a ³²P-labeled probe complementary to the NS2B-NS3 gene region of DEN-2 RNA. (a) RNA from H 9.1 cells. (b) RNA from FB 9.1 cells. (c) Relative amounts of DEN-2 RNA determined by phosphorimager analysis. dpi, days postinfection. *, data from 11-day-postinfection time point for H 9.1 cells were unavailable.

FIG. 8.

Northern blot hybridization to detect siRNA in mosquito cell lines. Total RNA was extracted from untransformed C6/36 cells (A), the H 9.1 cell line (B and D), and the FB 9.1 cell line (C and E) and enriched for small RNA (Materials and Methods). Lanes A to C were probed with ³²P-labeled strand-specific antisense RNA complementary to the DEN-2 prM gene; lanes D and E were probed with sense RNA from the DEN-2 prM gene.