Host-directed combinatorial RNAi improves inhibition of diverse strains of influenza A virus in human respiratory epithelial cells

<div><p>Influenza A virus infections are important causes of morbidity and mortality worldwide, and currently available prevention and treatment methods are suboptimal. In recent years, genome-wide investigations have revealed numerous host factors that are required for influenza to successfully complete its life cycle. However, only a select, small number of influenza strains were evaluated using this platform, and there was considerable variation in the genes identified across different investigations. In an effort to develop a universally efficacious therapeutic strategy with limited potential for the emergence of resistance, this study was performed to investigate the effect of combinatorial RNA interference (RNAi) on inhibiting the replication of diverse influenza A virus subtypes and strains. Candidate genes were selected for targeting based on the results of multiple previous independent genome-wide studies. The effect of single and combinatorial RNAi on the replication of 12 diverse influenza A viruses, including three strains isolated from birds and one strain isolated from seals, was then evaluated in primary normal human bronchial epithelial cells. After excluding overly toxic siRNA, two siRNA combinations were identified that reduced mean viral replication by greater than 79 percent in all mammalian strains, and greater than 68 percent in all avian strains. Host-directed combinatorial RNAi effectively prevents growth of a broad range of influenza virus strains <i>in vitro</i>, and is a potential therapeutic candidate for further development and future <i>in vivo</i> studies.</p></div

Inhibition of diverse influenza A virus in siRNA treated NHBE cells.

<p>The siRNA treatments listed on the left column were evaluated against each of twelve influenza strains noted across the top. Each combination was tested in triplicate and normalized to Allstars nontargeting siRNA. Relative mean viral growth is reported; 1.00 denotes no change relative to non-targeting siRNA, 0.50 denotes a 50% decrease in viral growth, and 0.00 denotes complete inhibition of viral growth.</p

Cytotoxicity of siRNA treatment in NHBE cells.

<p>Cell viability of NHBE cells at 0, 24, 48, and 72 hours post siRNA transfection. Values are normalized to mean OD of cells transfected with non-targeting siRNA at each corresponding time interval after transfection. Relative values below 0.7 are considered toxic; all other values are considered viable, with those shaded in dark grey having the greatest cell viability (>0.9) and those in light grey being near the cutoff for cell viability (0.7 to 0.79).</p

Description of influenza virus strains used.

<p>Description of influenza virus strains used.</p

siRNA knockdown of mRNA in NHBE and the impact on influenza A/WSN/1933 (WSN) replication.

<p>(a) Knockdown efficiency of siRNA in NHBE cells; the components within Combo1 are shown as a representative example. Bars represent mean (+ SEM) knockdown relative to non-targeting siRNA 24 hours post transfection. Knockdown data for additional combinations of targets is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197246#pone.0197246.s001" target="_blank">S1 Table</a>. (b) Inhibition of viral replication when genes were targeted singly, or in combination. For each bar, the targeted genes are shown (X) below. The Allstars negative control (N) and siNP positive control (P) are shown. Results are displayed as normalized to viral growth in cells transfected with non-targeting siRNA. (* p < 0.05, ** p < 0.01). NHBE cells were transfected with siRNA 24 hours after plating. Cells were infected with WSN virus (MOI = 0.2) 24 hours following siRNA transfection and incubated for 48 hours prior to evaluating viral titer by plaque assay.</p

siRNA knockdown of mRNA and protein expression of targeted genes in A549 cells and the impact on influenza A/WSN/1933 (WSN) replication.

<p>(a) Knockdown efficiency of mRNA in A549 cells. Bars represent mean (+ SEM) knockdown relative to non-targeting siRNA 24 hours post transfection. (b) and (c) Knockdown efficiency of protein in A549 cells. Lysates from siRNA- and mock-transfected A549 cells were quantified using the BCA protein assay, then 10 μg (COPA, predicted size of 140 kDa) or 5 μg (RPS14, predicted size of 17 kDa) were analyzed by SDS-PAGE as 2 biological replicates. The protein band intensities of COPA and RPS14 (green) were normalized to the levels of GAPDH (red). (d) Inhibition of viral replication when genes were targeted singly, or in combination. For each bar, the targeted genes are shown (X) below. The Allstars negative control (N) and siNP positive control (P) are shown. Results are displayed as normalized to viral growth in cells transfected with non-targeting siRNA. (* p < 0.05, ** p < 0.01). A549 cells were transfected with siRNA 24 hours after plating. Cells were infected with WSN virus (MOI = 0.12) 24 hours following siRNA transfection and incubated for 48 hours prior to evaluating viral titer by plaque assay.</p

Bahl et al H6 Dataset

Bahl et al H6 Datase

Bahl et al H9 Dataset

Bahl et al H9 Datase

Bahl et al H3 Dataset

Bahl et al H3 Datase

Mean migration rates between avian ecosystems across sampling locations.

<p>Statistically supported interactions are shown in bold. Italicized text indicates interactions between ecosystems.</p