Ablation of viral gene expression by dsRNA against RSV P mRNA
The RNA genome of RSV is about 15 kb long and contains 11 documented protein-coding genes [13]. Three viral proteins are minimally required to reconstitute the functional transcription complex of NNR viruses [3]: the nucleocapsid protein (N) that wraps the negative-strand genome RNA and its full-length complement, the positive-strand antigenome RNA, thus converting them into highly nuclease-resistant, chromatin-like templates; the large protein (L), which is the major subunit of the RdRP; and the phosphoprotein (P), which is the smaller subunit of RdRP and an essential transcription factor of L [19–22]. In RSV, optimal transcription, although not replication, additionally requires the transcription antitermination protein M2-1 [13]. In addition, cellular actin, and to a lesser extent, profilin, are also required for viral transcription [17, 18].
The overall steps of a NNR viral macromolecular synthesis in the infected cell are relevant for this paper, and are briefly described here [3]. The L protein is believed to encode the basic RNA polymerization function, and binds to the viral promoter at the 3' end of the genomic RNA to initiate transcription. However, the P protein is essential for the RdRP holoenzyme to exit the promoter and to form a closed complex that is capable of sustained elongation [20]. The preformed RdRP brought in by the infecting viral nucleocapsids catalyzes the first rounds of transcription, known as primary transcription. In its "transcription mode", the viral RdRP starts and stops at the beginning and end, respectively, of each viral gene, and this results in the synthesis of individual gene mRNAs. Unlike the full-length genomic and antigenomic RNA, the mRNAs are 5'-capped, 3'-polyadenylated, and do not bind N protein. Translation of these mRNAs results in de novo synthesis of viral proteins. The availability of large quantities of N protein then allows encapsidation of nascent leader RNA by N. This leads to the switching of the RdRP to the "replication mode", resulting in the synthesis of full-length, encapsidated anti-genomic RNA, which is in turn replicated into more genomic RNA [15]. Thus, the very requirement of N for replication ensures that all full-length genomic and antigenomic RNA are wrapped with N protein, i.e., encapsidated [15]. The new pool of replicated genomic RNA serves as templates for secondary transcription. It should be obvious from the foregoing that the de novo macromolecular synthesis accounts for the major burst of viral protein and RNA in the infected cell. Specifically, if the de novo synthesis of the essential subunits of viral RdRP – such as L or P – is inhibited, it will abolish the bulk of viral transcription and replication, and hence, viral translation [3].
To test the effectiveness of the anti-P dsRNA, we transfected the dsRNA into A549 cells, and infected the cells with RSV. Subsequently, the amount of intracellular P protein was directly monitored by immunoblot analysis using anti-P antibody. Results presented in Fig. 1 show a nearly 90% reduction of P protein using as little as 10 nM dsRNA. Although we have not tested lower amounts of dsRNA for P, the severe loss of P protein at 10 nM dsRNA and only a slightly greater loss with higher dsRNA concentrations (Fig. 1) suggest that it may be possible to cause substantial ablation of P protein at dsRNA concentrations even below 10 nM.
The phenotypic effect of loss of P was further examined by measuring progeny viral titer, overall viral protein synthesis, and syncytium formation, as described under Materials and Methods. Yield of progeny virus in 20 nM dsRNA-treated cells was found to be reduced by 10 fold, and was reduced by at least 104 fold at 100 and 300 nM dsRNA (data not shown). De novo viral protein synthesis was measured by metabolic labeling with S35-Met/Cys followed by immunoprecipitation. As shown in Fig. 1, all viral proteins detectable in the precipitate were drastically diminished in the dsRNA-treated cells, as would be expected in the event of a loss of the P protein. The inhibition of viral growth was further reflected in the essentially complete loss of cell fusion (syncytia) in the treated cells (Fig. 2). In fact, the RSV-infected anti-P dsRNA-treated cells were morphologically indistinguishable from control uninfected ones even at 5 days post-infection, which was the longest time period for which they were observed. The presence of equal amounts of actin in all the samples confirmed that the observed inhibition of viral proteins is not due to a general degradation of proteins.
The specificity of dsRNA activity was further tested by using a dsRNA against cellular lamin A/C that was earlier shown to specifically abrogate lamin A/C synthesis in a variety of cultured cell lines [2]. As shown in Fig. 1 (lane 'La'), the anti-lamin dsRNA, while abrogating lamin protein (data not shown) had no effect on RSV protein synthesis. Furthermore, a mismatched anti-P dsRNA in which the lowercase A-U base pair (see the dsRNA sequences in Materials and Methods) was altered to a G-C pair also failed to inhibit viral translation (data not shown), confirming that a perfect match is needed for the dsRNA effect, hence its extreme specificity of action.
Lack of syncytium in RSV-infected cells treated with anti-F dsRNA
Fusion of the infected cells is a hallmark of all Paramyxoviruses including RSV (as also in some other viruses, such as HIV), and the resultant mass of fused cells is referred as a syncytium, from which respiratory syncytial virus derives its middle name. The fusion protein F is by far the most important viral glycoprotein that is central to the cell fusion activity [29]. Since the P and F proteins have such diverse roles in viral life cycle, we decided to investigate the effect of dsRNA on F as a second test gene, and also to compare and contrast the two respective phenotypes.
First, to test the effectiveness of the anti-F dsRNA intracellularly, we probed the infected cell monolayer with anti-F antibody by indirect immunofluorescence (Fig. 3). Results clearly demonstrated the abundant synthesis of F protein as cytoplasmic fluorescence in cells that were not treated with dsRNA; the nuclei of the same cells could be visualized by staining with DAPI. In contrast, cells treated with just 3 nM anti-F dsRNA showed a substantial loss of F stain. At 20 nM dsRNA, F protein was undetectable.
Second, immunoblot analysis (Fig. 4, top panel) revealed that anti-F dsRNA, at concentrations as low as 20 nM, produced a severe reduction in F protein levels. Again, no effect was seen on cellular profilin, ruling out a general protein loss. The anti-F dsRNA also had no effect on P protein levels, suggesting that such dsRNAs do not activate a general antiviral response that might abrogate all viral mRNA translation. This was further corroborated by the direct measurement of de novo viral protein synthesis by metabolic labeling (Fig. 4, bottom panel). Results showed that the synthesis of F only was affected while all other viral proteins were translated in normal amounts, which is in agreement with the notion that F has little or no role in intracellular viral macromolecular synthesis.
Finally, the phenotype of F protein loss was tested by examining syncytia formation, and as presented in Fig. 2, no syncytia could be discerned in anti-F dsRNA-treated cells (Panel B). However, a cytopathic effect was still visible, which is most likely the result of intracellular replication of the virus. This demonstrates an interesting contrast with the anti-P dsRNA (Panel C), which inhibited all viral gene expression, and therefore, the resultant monolayer exhibited essentially the same appearance as the uninfected one (Panel D).
Direct measurement of intracellular F mRNA by semi-quantitative RT-PCR showed a nearly 15-fold loss caused by anti-F dsRNA (Fig. 5, top panel). Similar RT-PCR of viral genomic RNA, viral P mRNA, or cellular actin mRNA revealed essentially no reduction, suggesting that the dsRNA did not activate a general antiviral response, and did not directly target genomic RNA. Together, these results directly demonstrate that the dsRNAs promote ablation of the specific mRNA target, which most likely underlies the loss of the respective proteins.
Anti-RSV dsRNAs do not activate an interferon response
As mentioned before, cytoplasmic dsRNA can trigger a series of signaling reactions that lead to interferon (IFN) synthesis [5, 6]. In the "interferon response", dsRNA molecules activate protein kinase PKR and 2',5'-oligonucleotide synthetase. One of the effects of PKR is to phosphorylate the α subunit of the general translation initiation factor eIF-2, which constitutes a major mechanism for global translation arrest. The 2', 5'-oligonucleotide synthetase activates RNase L that in turn catalyses non-specific degradation of mRNA. Since NNR viruses co-opt the cellular translation machinery, the interferon response thus causes severe inhibition of viral translation. Interestingly, the IFN response requires long dsRNA [5, 6], and it has been conjectured that the improved specificity of the 21-nucleotide long dsRNA in cultured mammalian cells is probably due to their inability to activate the IFN response [2]. We provide several lines of direct and indirect evidence that the dsRNAs described here did not activate a general IFN response. First, the inhibitory effect of each dsRNA was gene-specific (Figs. 1, 2, 4). Second, growth of VSV is known to be highly sensitive to IFN, however, its replication in A549 cells was not affected by any of the dsRNAs described here (data not shown). Lastly, elevated phosphorylation of eIF-2α has been used as a diagnostic marker of IFN response [30]. We, therefore, examined the phosphorylation status of eIF-2α in A549 cells following transfection with these dsRNAs. Results (Fig. 5, bottom panel) showed no increase in phosphorylation. In positive controls, A549 cell treated with calcium mobilizers (thapsigargin or A23178) did increase eIF-2 phosphorylation, as has been shown earlier [30]. These results provide the first direct evidence that the 21-nt long double-stranded siRNAs fail to trigger interferon response in mammalian cells, and hence, can be used as specific antiviral agents.