Poly-ADP Ribosyl Polymerase 1 (PARP1) Regulates Influenza A Virus Polymerase

Figure 3

Inhibitor of PARP1/2 proteins PARylation activity increases IAV RDRP activity. (a) Assay of cellular PARylation after treatment by 4-amino-1,8-naphthalimide (4-AN) for 20h in A549 cells. (b) Poly-ADP ribosylation (PARP) enzymatic activity was analyzed in protein lysates from A549 cells treated with DMSO vehicle, 20uM 4-AN, or 1uM doxorubicin (DOXO), and infected 20h with IAV (A/PR/8/34 H1N1, MOI = 1); lysates were analyzed by PARylation assay. (c) HEK 293T cells were transfected with NP and polymerase cDNA plasmids in IAV minigenome reporter assay and untreated, treated with vehicle (DMSO), or increasing doses of PARP inhibitor drug (4-AN). (d) HEK 293T cells were transfected with RdRP firefly luciferase reporter construct one day prior vehicle (DMSO) or treatment with 25uM 4-AN and infected with low-path, H5N1 HALo virus (MOI = 1). Infected cell viral polymerase activity was analyzed after 20h by luciferase assay; two-tailed t-test () p=0.06. (e) Single-step growth of H5N1 HALo virus (MOI = 1) in A549 cells pretreated with 4-AN or vehicle (DMSO) as indicated. Infectious titer (IU) released 1d.p.i. measured by limiting dilution assay for NP-positive cells. Two-tailed t-test for significance in comparison to vehicle: , p<0.05; ns, not significant. All experiments were performed in biological duplicates with two readings per well.

(a) Uninfected A549 cells

(b) Influenza infected A549 cells

(c) Influenza minigenome reporter

(d)

(e)