Influenza virus segment 5 (+)RNA - secondary structure and new targets for antiviral strategies

Figure 1. Self-folding of (+)RNA5 predicted by RNAstructure 5.5 using experimental data obtained in 23 °C as constraints. Strong DMS, CMCT and kethoxal modifications, as well as SHAPE reactivities converted to pseudo-free energies were used. Additionally medium reactivity DMS, CMCT, kethoxal and results from RNase H cleavage in presence of DNA oligonucleotides (neither used in modeling) are also annotated. Red lines indicate regions of structure that differ at higher temperature (37 °C). The numbering of (+)RNA5 is from its 5′ end. The AUG start codon spans nucleotides 46–48.

Figure 2. Chemical modifications data at 23 °C with NMIA for (+)RNA5 detected by reverse transcription with labeled primers followed by capillary electrophoresis. (A) Example of capillary electrophoresis raw data for nt 311–222 showing modified RNA (dark green line), unmodified control (light green line) and dideoxy ladders (C – red line and U – black line). (B) (+)RNA5 nt reactivities from SHAPE for the same region showed in panel A, in reverse order (222–311 nt) on the graph. (C) (+)RNA5 nt reactivities for SHAPE across the entire (+)RNA5. The nt region (222–311) showed on panel B is marked by line.

Figure 3. Predicted probability of nt being paired or single stranded in (+)RNA5 using RNAstructure program. Probability lower than 50% is not colored. The partition function calculation incorporated restraints from strong reactivity of DMS, CMCT, kethoxal and SHAPE (converted to pseudo-energy).

Figure 4. Conservation of (+)RNA5 secondary structure in type A of influenza viruses. Colors indicate percentage of canonical base pairing preserved across influenza A sequences for (+)RNA segment 5.

Figure 5. (+)RNA5 accessibility to oligonucleotides. Results from microarray mapping and RNase H cleavage in presence of DNA oligonucleotides at 37 °C are annotated. Additionally, target regions for designed ASOs are showed in the context of (+)RNA5 structure. Binding sites of pentamer probes are denoted by the middle nucleotide of the five nucleotides complementary in the RNA. In case of hexamer probes, binding sites are denoted by the fourth complementary nt in the RNA. Site 264 binds a heptamer and the middle nt of the seven nt complementary in the RNA is marked. The red line indicates regions of structure difference upon higher temperature (37 °C).

Figure 6. Antiviral activity of 750 nM ASOs in MDCK cells for A/California/04/2009 after 24 h postinfection. Antiviral activity was analyzed by immunofocus assay. The mean was calculated from three independent experiments, each containing three technical repeats and the standard deviation is shown. C - MDCK cells infected by virus, L - MDCK cells treated with Lipofectamine 2000 and infected, Neg 5 – MDCK cells transfected with control ASO Neg 5 and infected, N - MDCK cells transfected with control ASO N and infected. The remaining labels indicate MDCK cells transfected with indicated influenza A targeting ASOs and infected. Statistics for comparison to sample L were calculated using a two-tailed T-test (p < 0.01).

Figure 7. Antiviral activity of 750 nM ASOs in MDCK cells for A/California/04/2009 after 24 h postinfection. Antiviral activity was analyzed by qRT-PCR. The mean was calculated from three independent experiments each containing three technical repeats, each repeat was analyzed by three independent qRT-PCR reactions and the standard deviation is shown. C - MDCK cells infected by virus, L - MDCK cells treated with Lipofectamine 2000 and infected, Neg 5 – MDCK cells transfected with control ASO Neg 5 and infected, N - MDCK cells transfected with control ASO N and infected. The remaining labels indicate MDCK cells transfected with indicated influenza A targeting ASOs and infected. *Statistics for comparison to sample L were calculated using a two-tailed T-test (p < 0.01).