Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

Abstract

Background: Post-transcriptional gene silencing (PTGS) by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs) for their ability to silence cytoplasmic RNA genomes.

Results: Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected.

Conclusions: Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Figure 1

Ablation of RSV P protein by anti-P dsRNA. Transfection of A549 cells with 0 (no dsRNA), 10, 100, or 300 nM dsRNA and infection with RSV were carried out as described under Materials and Methods. Lane 'U' indicates control, uninfected cells. Top: Immunoblot (Western) of total cell extracts was performed with either rabbit anti-P or monoclonal anti-actin antibody (Boehringer-Mannheim), as indicated. Bottom: Viral protein synthesis in dsRNA-treated cells was measured by standard immunoprecipitation procedures as described previously [17]. Infected A549 cells (or uninfected control, lane 'U') were metabolically labeled with ³⁵S-(methionine plus cysteine) at 18 h post-infection, followed by lysis of the cells, precipitation with anti-RSV antibody, and analysis of the labeled proteins by SDS-PAGE and autoradiography. 'La' represents treatment with 100 nM anti-lamin A/C dsRNA. The different viral protein bands are so indicated.

Figure 2

Effect of dsRNA on the cell fusion activity of RSV. A549 monolayers were transfected with 20 nM of anti-P or anti-F dsRNA and infected with RSV as described under Materials and Methods. At 40 h p.i., the monolayers were examined under a Nikon TS100F phase-contrast microscope at 40× magnification and digitally photographed with a Nikon Coolpix 995 camera. Note the syncytia in 'A', cytopathic effect without syncytia in 'B', and monolayers that appear unaffected and identical in 'C' and 'D'.

Figure 3

Ablation of RSV F by anti-F dsRNA. Anti-F dsRNA, RSV infection, and immunostaining of A549 monolayer were performed as described under Materials and Methods. Right panel shows the nuclear staining of the same cells using DAPI (Blue). Note the substantial reduction of F (Green) with as low as 3 nM anti-F dsRNA, and reduction to background levels by 20 nM dsRNA.

Figure 4

Specificity of anti-F dsRNA. Experiments were done essentially as described for Fig. 2. A549 cells were transfected with the indicated amounts of anti-F dsRNA followed by infection by RSV as described under Materials and Methods. Top: Immunoblot of total cell extracts to detect RSV F, RSV P, and profilin; Bottom: Autoradiograph showing immunoprecipitated metabolically ³⁵S-labeled RSV proteins. 'U' represents uninfected cells. Note the specific loss of F protein, but no effect on other proteins.

Figure 5

Induction of target mRNA degradation but not interferon response by dsRNA. Panel A: Semi-quantitative RT-PCR to measure the indicated RSV gene mRNA and genomic RNA in A549 cells were performed as described under Materials and Methods. Where indicated (labeled '+'), anti-F dsRNA was used at a concentration of 20 nM. PCR samples were taken at the end of the number of cycles indicated on top (20, 22, 24, and 26). Actin mRNA was also quantitated as a control. Note that in dsRNA-untreated cells (labeled '-') the F band is visible even at 20 cycles, whereas in the treated cells, appearance of a comparable intensity required 4 additional PCR cycles, i.e., 16-fold more amplification. Panel B: Assay of eIF-2α phosphorylation. Metabolic ³²P-labeling and immunoprecipitation (IP) analysis of eIF-2 have been described in Materials and Methods. An autoradiograph of the gel is shown. The cells were treated with no RNA (lane C), 100 nM thapsigargin (lane 1), 100 nM A23178 (lane 2), 50 nM anti-P dsRNA (lane 3), or 50 nM anti-F dsRNA (lane 4). Note the increased phosphorylation of eIF-2 in lanes 1 and 2 only. The immunoblot (IB) shows that the total amount of eIF-2α protein was not affected by the treatments.