Oxysterol-binding protein family I is the target of minor enviroxime-like compounds

Abstract

Enviroxime is an antipicornavirus compound that targets host phosphatidylinositol 4-kinase III beta (PI4KB) activity for its antipicornavirus activity. To date, several antipoliovirus (PV) compounds similar to enviroxime that are associated with a common resistance mutation in viral protein 3A (a G5318A [3A-Ala70Thr] mutation in PV) have been identified. Most of these compounds have a direct inhibitory effect on PI4KB activity, as well as enviroxime (designated major enviroxime-like compounds). However, one of the compounds, AN-12-H5, showed no inhibitory effect on PI4KB and was considered to belong to another group of enviroxime-like compounds (designated minor enviroxime-like compounds). In the present study, we performed a small interfering RNA (siRNA) sensitization assay targeting PI4KB-related genes and identified oxysterol-binding protein (OSBP) as a target of minor enviroxime-like compounds. Knockdown of OSBP and OSBP2 increased the anti-PV activities of AN-12-H5 and a newly identified minor enviroxime-like compound, T-00127-HEV2, and also to T-00127-HEV1 to a minor extent, in the cells. A ligand of OSBP, 25-hydroxycholesterol (25-HC), acted as a minor enviroxime-like compound. Minor enviroxime-like compounds induced relocalization of OSBP to the Golgi apparatus in cells. Treatment of the cells with major or minor enviroxime-like compounds suppressed the expression of genes (HMGCS1 and SQLE) in the SREBP/SCAP regulatory pathway and diminished endogenous phosphatidylinositol 4-phosphate (PI4P) at the Golgi apparatus. Our results suggested that minor enviroxime-like compounds are phenotypically identical to 25-HC and that major and minor enviroxime-like compounds suppress the production and/or accumulation of PI4P in PV-infected cells by targeting PI4KB and OSBP family I activities, respectively.

Fig 1

(A) Characterization of T-00127-HEV2. (Top) Structure of T-00127-HEV2. (Bottom) Inhibitory effect of T-00127-HEV2 on PV pseudovirus and viability of RD cells. PV pseudovirus infection (luciferase assay), viability of cells, or PV1(Mahoney) infection (number of copies of the viral genome at 7 h p.i. in RD cells) in the absence of compounds was taken as 100%. T-00127-HEV2 showed precipitation above 50 μM. (B) Specificity of resistance mutations to T-00127-HEV2. RD cells were infected with PV pseudovirus mutants that have resistance mutations to GuHCl (U4614A), brefeldin A (G4361A plus C5190U), and enviroxime (G5318A) in the presence of antienterovirus compounds: T-00127-HEV1 (3.1 μM), AN-12-H5 (25 μM), and T-00127-HEV2 (6.3 μM). Relative PV pseudovirus infection is shown, where parental PV pseudovirus infection in the presence of each compound was taken as 1. n = 3. (C) Inhibitory effects of enviroxime-like compounds on in vitro activity of PI4KB and PI4KA. In vitro kinase activities were analyzed in the presence of each compound (1 μM) and ATP (10 μM). (D) HCV replicon replication in the presence of enviroxime-like compounds. HCV replicon replication in the absence of compounds was taken as 100%. The error bars indicate standard deviations.

Fig 2

siRNA sensitization assay for enviroxime-like compounds targeting PI4KB binding proteins and PI4P binding proteins. (A and B) Normalized PV pseudovirus infection in the presence of AN-12-H5 (1.6 and 0.78 μM) or T-00127-HEV1 (0.78 μM) and net PV pseudovirus infection in siRNA-transfected HEK293 cells targeting PI4KB binding proteins and PI4P binding proteins (A) or OSBP-related proteins (B). Normalized PV pseudovirus infection is the ratio of PV pseudovirus infection in compound-treated and siRNA-transfected cells (percent) to PV pseudovirus infection in siRNA-transfected cells in the absence of compounds (percent). Normalized PV pseudovirus infection in mock-transfected cells was taken as 1. n = 3. The error bars indicate standard deviations.

Fig 3

(A) Characterization of 25-HC. (Top) Structure of 25-HC. (Bottom) Inhibitory effect of 25-HC on PV pseudovirus and viability of RD cells. PV pseudovirus infection or viability of the cells in the absence of compounds was taken as 100%. 25-HC showed precipitation above 13 μM. (Right) Specificity of resistance mutations to 25-HC. RD cells were infected with PV pseudovirus mutants that have resistance mutations to GuHCl (U4614A), brefeldin A (G4361A plus C5190U), and enviroxime (G5318A) in the presence of 25-HC (25 and 50 μM). Relative PV pseudovirus infection is shown, where parental PV pseudovirus infection in the presence of 25-HC was taken as 1. n = 3. (B) siRNA sensitization assay for enviroxime-like compounds. HEK293 cells were transfected with siRNA targeting PI4KB or OSBP or with nontargeting siRNAs (nontargeting siRNAs 1 and 2), and the sensitization effect was analyzed in the presence of enviroxime-like compounds (T-00127-HEV1 [0.78 μM], enviroxime [0.16 μM], AN-12-H5 [1.6 μM], T-00127-HEV2 [0.78 μM], and 25-HC [3.1 μM]) and with GuHCl (125 μM) as a control compound. Normalized PV pseudovirus infection is shown, with P values. n = 4. The error bars indicate standard deviations.

Fig 4

Effects of minor enviroxime-like compounds on OSBP relocalization and gene expression in the SREBP/SCAP regulatory pathway. (A) Effects of enviroxime-like compounds on relocalization of OSBP. HEK293 cells expressing OSBP-EGFP or CERT-EGFP were treated with 10 μM T-00127-HEV1, AN-12-H5, T-00127-HEV2, or 25-HC or with 2 mM GuHCl for 1 h. Scale bars, 20 μm. (B) Effects of enviroxime-like compounds on expression of HMGCS1 and SQLE mRNAs. RD cells were treated with 10 μM T-00127-HEV1, AN-12-H5, T-00127-HEV2, or 25-HC or with 2 mM GuHCl for 6 h. Total RNA was collected from the treated cells and subjected to quantitative real-time RT-PCR. Relative expression levels of ACTB, HMGCS1, and SQLE mRNAs normalized by GAPDH mRNA are shown. n = 3. (C) Evaluation of the effective period for addition of enviroxime-like compounds in PV infection. Enviroxime-like compounds (20 μM) and GuHCl (2 mM) were added to RD cells at the indicated times (−3 h to 6 h p.i.). PV pseudovirus was added at 0 h p.i., and the luciferase activity was measured at 7 h p.i. PV pseudovirus infection in the absence of compounds was taken as 100%. The times of drug addition, where significant suppression of PV pseudovirus infection was observed (P < 0.01), are indicated by lines. n = 3. The error bars indicate standard deviations.

Fig 5

Effects of enviroxime-like compounds on PI4P accumulation at the Golgi apparatus. Indirect immunofluorescence of PI4KB and PI4P in RD cells treated with 10 μM T-00127-HEV1, AN-12-H5, T-00127-HEV2, 25-HC, or 2 mM GuHCl overnight is shown. Blue, nuclear (staining with Hoechst 33342); green, PI4KB; red, PI4P. Scale bars, 10 μm. The white arrows indicate some of the colocalized sites, and the cyan arrows indicate some of the noncolocalized sites.