Inhibition of HAZV replication using segment-specific siRNAs
To evaluate the inhibitory activity of siRNAs on HAZV replication, twelve siRNAs were designed to target the mRNAs produced by the L, M, and S genomic segments. We analyzed the inhibitory effect of the L, M and S-specific siRNAs. As shown in figure 1A, when used at 100 nM, siS1, siS2 and siS3 strongly inhibited virus replication by 87.8%, 91.6% and 86.1%, respectively, compared to siNT control (p < 0.05). In contrast, siS4 did not induce any significant antiviral effect in A549-infected cells. siM1, siM2 and siM3 showed moderate but significant activities on HAZV replication (38.3%, 56.2% and 29.4% of inhibition respectively) whereas siM4 did not have any activity (figure 1B). Lastly, siL2, siL3 and siL4 did not inhibit HAZV replication (p > 0.05) while siL1 slightly did.
Inhibition of HAZV replication by siS1 and siS2 in A549 cells
Since siS1 and siS2 showed the most efficient anti HAZV activity, we decided to further focus on the characterization of these two siRNAs. We first tested their efficacy at 100 nM in cells infected at different multiplicity of infection (i.e. MOI 0.01, 0.1 and 1). Whatever the viral load, a significant inhibition of HAZV replication was observed for both siRNAs when compared to siNT: the inhibition ranged from 83.1% to 97.1% for siS1 and from 94.4% to 98.3% for siS2.
Because siRNAs treatment could be cytotoxic and therefore may affect viral growth, we performed a Trypan blue exclusion assay to evaluate cell growth and viability upon siRNA transfection. The morphology of treated cells was examined daily using phase-contrast light microscopy. Three days post siRNA transfections, the cell number per well was determined and compared to non treated cells by manual counting with a hematocytometer. In these experiments, we did not detect any cytotoxic effect of siS1 and siS2 on A549 cells at any concentrations used (not shown). We also investigated whether the IFN pathway could be stimulated in siRNAs transfected cells as reported in earlier studies. We found that the siRNAs tested did not induce any IFN-β response whereas the poly(I:C) control was a good stimulator (not shown). Thus, HAZV inhibition was siS1 and siS2 specific and was not due to any side effects.
We then examined the antiviral effects of increasing concentrations of siS1 and siS2. A concentration dependent inhibitory activity was observed for both siRNAs when compared to siNT. siS1 induced an average inhibition of 64.2% at 0.1 nM and 86% at 100 nM. The production of infectious HAZV particles was reduced by 70.6% at 0.1 nM and 94% at 100 nM when cells were transfected with siS2. No significant viral inhibition was observed at siRNAs concentration of 0.01 nM. From these experiments the estimated 50% effective concentration (EC50) was 0.09 nM for siS1 and 0.07 nM for siS2. Thus, both siRNAs efficiently inhibited HAZV replication in a concentration dependant manner, in cell culture.