Figure 3. Hsp inhibitor KNK437 impairs IAV polymerase function, viral protein accumulation and overall replication.

(a) KNK437 significantly reduces RNP activity. A549 cells were transfected with plasmids containing PA, PB1, PB2, NP, NS1-Renilla luc/pPolI derived from A/Puerto Rico/8/1934 H1N1 along with internal transfection control plasmid pGL3 constitutively expressing Firefly luciferase. Cells were treated with 200 μM KNK437 at the time of transfection and relative IAV Pol activity was measured by taking ratio renilla/firefly luc counts. (b) KNK437 mediated Hsp inhibition reduces NP mRNA and vRNA levels. A549 cells were pretreated with DMSO or 200 μM KNK437 for 6 h followed by infection with IAV WSN at MOI = 5. NP mRNA and vRNA levels were quantified by qRT-PCR at 4 h p.i. in DMSO or KNK437 treated cells. The fold change in levels was calculated with reference to control infected cells. (c) Hsp inhibition significantly impairs viral protein accumulation. A549 cells were pretreated with DMSO or 200 μM KNK437 for 6 h followed by infection with PR8 virus at MOI = 5. Cells were harvested at 4 h and 8 h p.i. and cell lysate was subjected to SDS-PAGE followed by western blotting to check viral protein levels. (d) Densitometry analysis was done using ImageJ and NP levels normalized against actin were plotted on the graph. (e) KNK significantly impairs virus replication. Supernatant (cell media) was collected at 24 h p.i. to perform plaque assay. Data show mean ± S.D. from one representative experiment (n = 3) of at least three independent experiments. Statistical significance was determined using Student’s t test. *p < 0.05.