Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity

Abstract

Enviroxime is an antienterovirus compound that targets viral protein 3A and/or 3AB and suppresses a step in enterovirus replication by unknown mechanism. To date, four antienterovirus compounds, i.e., GW5074, Flt3 inhibitor II, TTP-8307, and AN-12-H5, are known to have similar mutations in the 3A protein-encoding region causing resistance to enviroxime (a G5318A [3A-Ala70Thr] mutation in poliovirus [PV]) and are considered enviroxime-like compounds. Recently, antienterovirus activity of a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor, PIK93, was reported, suggesting that PI4KB is an important host factor targetable by antienterovirus compounds (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we analyzed the inhibitory effects of previously identified enviroxime-like compounds (GW5074 and AN-12-H5) and a newly identified antienterovirus compound, T-00127-HEV1, on phosphoinositide (PI) kinases. We found that T-00127-HEV1 inhibited PI4KB activity with a higher specificity for than other PI kinases, in contrast to GW5074, which had a broad specificity for PI kinases. In contrast, AN-12-H5 showed no inhibitory effect on PI4KB activity and only moderate inhibitory effects on PI 3-kinase activity. Small interfering RNA (siRNA) screening targeting PI kinases identified PI4KB is a target of GW5074 and T-00127-HEV1, but not of AN-12-H5, for anti-PV activity. Interestingly, T-00127-HEV1 and GW5074 did not inhibit hepatitis C virus (HCV) replication, in contrast to a strong inhibitory effect of AN-12-H5. These results suggested that PI4KB is an enterovirus-specific host factor required for the replication process and targeted by some enviroxime-like compounds (T-00127-HEV1 and GW5074) and that enviroxime-like compounds may have targets other than PI kinases for their antiviral effect.

FIG. 1.

Characterization of T-00127-HEV1 and inhibitory effects on PI kinases. (A) Characterization of T-00127-HEV1. Upper panel, structure of T-00127-HEV1. Lower panels, inhibitory effect of T-00127-HEV1 on PV pseudovirus (left panel) and viability of RD cells (right panel). PV pseudovirus infection in the absence of compounds was taken as 100%. (B) Inhibitory effect of T-00127-HEV1 on enterovirus infection. RD cells were infected with PV1(Mahoney), EV71(Nagoya), or CVB3(Nancy) at MOI of 10, 1.0, and 0.1 in the absence of T-00127-HEV1 and then were treated with 0 or 10 μM T-00127-HEV1 from 1 h p.i. The total number of copies of viral genomes in the cells at 16 h p.i. is shown. (C) Specificity of mutations causing resistance to antienterovirus compounds. RD cells were infected with PV pseudovirus mutants that have mutations causing resistance to GuHCl (U4614A), brefeldin A (G4361A plus C5190U), and enviroxime (G5318A) in the presence of antienterovirus compounds GW5074 (25 μM), PIK93 (1.3 μM), and T-00127-HEV1 (4.0 μM). PV pseudovirus infection in the absence of compounds was taken as 100%. (D) Inhibitory effects of enviroxime-like compounds on in vitro activities of PI kinases. Upper panel, in vitro activities of PI kinases were analyzed in the presence of enviroxime-like compounds (GW5074, AN-12-H5, and T-00127-HEV1 at a concentration of 10 μM) with an ATP concentration of 10 μM. Lower panel, inhibitory effect of T-00127-HEV1 on the in vitro PI4KB activity measured with an ATP concentration of 10 μM.

FIG. 2.

Effect of siRNA treatment targeting PI kinases on PV pseudovirus infection. (A) Effect of siRNA treatment on cell viability. The viability of mock-treated cells was taken as 100%. (B) Effect of siRNA treatment on PV pseudovirus infection. PV pseudovirus infection in mock-transfected cells was taken as 100%. (C) Net PV pseudovirus infection in siRNA-treated cells. Net PV pseudovirus infection is the ratio of PV pseudovirus infection in siRNA-transfected cells (percent) to cell viability (percent). The net PV pseudovirus infection in mock-transfected cells was 1. (D) Western blot analysis of cell lysates of the cells transfected with siRNAs targeting the GBF1, ARF1, and PI4KB genes.

FIG. 3.

Effects of enviroxime-like compounds on PV pseudovirus infection in siRNA-transfected cells. (A) Normalized PV pseudovirus infection in the presence of GW5074 (3.1 μM), AN-12-H5 (1.5 μM), and T-00127-HEV1 (0.78 μM). Normalized PV pseudovirus infection is the ratio of PV pseudovirus infection in drug-treated and siRNA-transfected cells (percent) to PV pseudovirus infection in siRNA-transfected cells in the absence of compounds (percent). The normalized PV pseudovirus infection in mock-transfected cells was 1. (B) Normalized PV infection in the presence of GW5074, AN-12-H5, and T-00127-HEV1 at lower concentrations (1.5, 0.78, and 0.39 μM).

FIG. 4.

Effect of enviroxime-like compounds on PV pseudovirus infection in PI4KB siRNA-transfected cells. HEK293 cells were transfected with siRNA targeting the PI4KB gene or with nontargeting siRNAs (nontargeting siRNAs 1 and 2), and then sensitization of the siRNA-transfected cells was analyzed in the presence of GW5074 (3.1 μM), AN-12-H5 (1.5 μM), and T-00127-HEV1 (0.78 μM). *, P < 0.05.

FIG. 5.

Effects of enviroxime-like compounds on PV pseudovirus infection and HCV replicon replication. Inhibitory effects of enviroxime-like compounds on PV pseudovirus infection (left panel) and HCV replicon replication (right panel) are shown. PV pseudovirus infection or HCV replication in the absence of compounds was taken as 100%.