Figure 6. Over-expression of Hsp40/DnaJB1 promotes nuclear accumulation of IAV NP and enhances IAV replication.

(a) IAV NP nuclear distribution increases on up-regulating Hsp40/DnaJB1 levels. A549 cells were transfected with GFP or Hsp40-GFP. 24 h post-transfection, cells were infected with PR8 virus at MOI = 5. Cells were harvested 4 h post-infection, subjected to cellular fractionation followed by western blotting. HDAC1 and GAPDH were used as loading control for nuclear and cytoplasmic fractions respectively. (b) Over-expression of Hsp40 enhances the nuclear localization of transiently expressed NP. HEK293 cells were co-transfected with GFP or Hsp40-GFP and NP-pCDNA. 48 h post-transfection, cells were harvested and subjected to cellular fractionation followed by western blotting. SP1 and GAPDH were used as loading control for nuclear and cytoplasmic fractions respectively. (c) Over-expression of Hsp40 significantly enhances RNP activity. A549 cells were transfected with plasmids containing PA, PB1, PB2, NP and NS1-luc/pPolI derived from A/Puerto Rico/8/1934 H1N1 and internal transfection control plasmid pGL3 along with GFP or Hsp40-GFP. Cells were harvested 24 h post-transfection and effect on polymerase activity was determined by measuring change in luciferase activity. (d–g) A549 cells were transfected with control plasmid or Hsp40-GFP. 24 h post-transfection, cells were infected with virus at MOI = 5 (4 and 8 h) and 1 (24 h). Cells were harvested at different time points post-infection to perform western blotting (d,e) and qRT-PCR to check NP mRNA and vRNA levels at 4 h p.i. (f). Cell media was collected at 24 h p.i. to carry out plaque assay (g). The fold change in NP mRNA or vRNA levels and relative protein levels were calculated with reference to GFP transfected cells. 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.