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. 2002 Dec;76(24):12925-33.
doi: 10.1128/jvi.76.24.12925-12933.2002.

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

Zach N Adelman  1 , Irma Sanchez-Vargas, Emily A Travanty, Jon O Carlson, Barry J Beaty, Carol D Blair, Ken E Olson
Affiliations

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

Zach N Adelman et al. J Virol. 2002 Dec.

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.

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Figures

FIG. 1.
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.
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.
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.
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.
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.
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.
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.
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.
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