Initially, when we selected the targeting sequences within the CVB3 RNA, the 5′UTR and the initiation codon regions were chosen for potential weaker target controls because the UTR-binding proteins and/or translation initiation complexes may interfere with binding of siRNA and the RISC in these regions (http://www.rockefeller.edu/labheads/tuschl/sirna.html
). As expected, cells pretreated with siRNA-1 and -2 targeting the 5′UTR and initiation codon region demonstrated poorer antiviral capability than those pretreated with siRNA-3, -4, and -5 targeting the coding regions. However, several studies have demonstrated excellent protection against various virus infections by specific siRNAs targeting the 5′UTR (24
). These studies suggest that whether the binding of protein factors at certain sites of the 5′UTR has a negative or positive effect on siRNA function may depend on the local sequence characteristics. For certain regions, protein complex binding may change the higher ordered structure of the target sequence and, therefore, facilitate the access of the siRNA to the target sequence. On the other hand, if the siRNA and the protein factor compete for the same target, which is the regulatory sequence of gene expression, the binding of the protein factors may inhibit the RNAi activity of siRNA.
Among the siRNAs that we tested, siRNA-4, which is directed against the viral protease 2A region, was the most effective one, followed by siRNA-3, -5, -1, and -2. These results may be due to different positional accessibility caused by steric hindrance by a secondary or tertiary structure and/or protein binding. In this regard, there are inconsistent reports. Several studies focusing on the relationship between secondary structure and siRNA effects showed that the secondary structure at least in part influences the efficiency of siRNAs (22
). Conversely, it has been reported that the secondary structure of the target mRNA does not appear to have a strong effect on gene silencing (22
). Whether secondary structure plays a role in siRNA machinery binding is still debated. However, we believe that binding of cellular proteins on the target sites or the secondary structure of the mRNA may affect, at least in part, the efficacies of siRNAs in the cells. Although we are not clear on the higher ordered structure of the siRNA-4 targeting sequence in the cellular environment, we believe that it possesses a more accessible conformation for siRNA. This may also be related to the thermodynamic instability of the dsRNA intermediate at this specific locus during CVB3 transcription, which is evidenced by this segment lacking any continuous two repeating bases at the middle region (Table ), or this region may be favorable for binding of RISC required for siRNA function. In addition to the secondary structure and protein binding, the inefficacy of siRNA-2 may also be due to the sequence containing three continuous cytosines, which may hyperstack and therefore form agglomerates that potentially interfere in the silencing mechanism. Similarly, the siRNA containing five continuous guanosines targeted on the 5′UTR of hepatitis C virus did not show inhibitory ability on virus replication (51
). Therefore, avoiding more than three Gs or Cs in the siRNA may exclude this problem. Interestingly, the sequence covering the initiation codon region has been reported to be an effective target for gene knockdown by an antisense deoxynucleotide (AS-DON) agent (45
). Our previous study of antiviral activity of AS-DONs also confirmed this in anti-CVB3 replication (45
). This inconsistent data regarding siRNA targeting the same region may be due to the distinct mechanism of action for AS-DON. A commonly exploited antisense mechanism is RNase H-dependent degradation of the targeted RNA through recognition of a DNA-RNA heteroduplex (28
), while siRNAs bind to targeted RNA by Watson-Crick base pairing and induce site-specific cleavage of the RNAs by a specific unknown RNase. This suggests that the best target sequences for AS-DON may not be the best candidate sites for siRNAs. Due to the different targeting sites used for siRNA and AS-DON, it is hard to compare their inhibitory effects on CVB3 replication. Overall, under optimal conditions it seems that CVB3-specific siRNAs are more effective than AS-ODNs in terms of potency, efficacy, and duration, which is consistent with other studies (2
Sequence specificity of siRNA is very stringent, as single base pair mismatches between the siRNA and its target sequence dramatically reduce the silencing capability (3
). However, there are different reports on this issue with different experimental systems. A detailed siRNA functional anatomy analysis revealed that RNAi required a perfect match between cellular mRNA and the antisense strand of siRNA, but several mutations in the sense strand of siRNAs did not eliminate the gene silencing (14
). On the other hand, in another report an siRNA with two nucleotide mismatches in the central region still had partial inhibitory activity (51
). The experiments reported here used a positive single-stranded RNA virus which can produce a dsRNA intermediate during replication. This raises the possibility that siRNA may target positive, negative, or both strands of virus RNA. To clarify this question, we perform evaluations using a series of siRNA-4 mutants containing point mutations within the sense and/or antisense strands at different locations. The data suggest that only one point mutation in the middle of the antisense strand could eliminate the anti-CVB3 activity, whereas the corresponding mutation on the sense strand did not interfere with the viral replication, suggesting that the negative-strand RNAs produced during viral replication are not the direct target of siRNA. This result might be explained by the fact that the replicating negative strands of virus only exist as a double-stranded form in the vesicles (15
), thus, they are less likely to be accessible to siRNAs. Conversely, the positive strand is the recognition site for RNAi, which forms complementary base pairs with the antisense strand of the siRNA. This conclusion is not only drawn from our study using positive single-stranded RNA virus but also has been reported recently for a negative single-stranded influenza virus in which the siRNA targets the mRNA of virus during replication (17
). For the point mutation closest to the 5′ or 3′ end of the antisense siRNA, it is likely that the mismatch near the 5′ end has more negative effects on gene silencing than that near the 3′ end, which is consistent with a previous report (40
). However, the molecular mechanism of this phenomenon needs to be further studied.
Cotransfection of cells with two or more siRNAs targeting different sites on HIV-1 coreceptor CXCR4 mRNA has been reported to result in enhanced gene silencing compared to that of each single siRNA (25
). This could be explained by specific binding of certain siRNAs that may change the secondary structure of RNA and result in more accessible sites for other siRNA molecules. However, as with a previous report (22
), we did not observe enhancement effects when using any combinations of two agents, including siRNA-4, in our system. These particular siRNAs probably could not affect the secondary structure of the targets or open more space to other siRNAs, or the amount of siRNA-associated proteins was limited for silencing rather than target accessibility. In general, the reasons for the discrepancy between the studies may be due to differences in mRNA targets and the evaluation methods. For antiviral evaluation, although the underlying mechanism of the enhanced gene silencing with multiple specific siRNAs is not clear, cotransfection with multiple siRNAs may benefit long-term treatment, as mutated virus variants may be produced following infection to escape from protection by siRNA (3
To investigate whether escaping CVB3 mutants were generated following siRNA-4 treatment, a series of passages of CVB3 were challenged with fresh siRNA-4. However, we did not detect any mutants in this study. The discrepancy between previous reports (4
) and our result could be due to the fact that (i) exposure time to siRNA was not long enough compared to that of a previous HIV study, which showed emergence of mutation at the target site at 25 days posttreatment (4
); and (ii) a different targeting region was used, as the siRNA-4 targeting sequence is a critical site for CVB3, because mutations in this area would markedly impair the fitness of the virus. Therefore, highly conserved regions should be used as targets for siRNA design to limit the occurrence of escape mutants.
In summary, this in vitro study is the first step to demonstrate that siRNA technology is a very promising approach to antiviral gene therapy. The very strong anti-CVB3 activity of siRNA-4 has indicated attractive new directions for further investigation of the underlying mechanism and the development of siRNA-4 as a prophylaxis and therapy for CVB3 infection.