Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

Abstract

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.

Figure 1

CVB3 3A mutants are resistant to enviroxime, GW5074, and PI4KIIIβ inhibitor PIK93. (A, B) BGM cells were infected with CVB3 wt, CVB3 3A-V45A, or CVB3 3A-H57Y (A) or transfected with the corresponding RNA transcripts of full length infectious CVB3 clones (B). Immediately after infection or transfection, enviroxime, GW5074, or PIK93 were added to the cells. After 8 h, cells were lysed by freeze-thawing to release intracellular virus particles and the total virus titer was determined by endpoint titration. (C) BGM or HeLa cells were incubated with the indicated compounds for 8 h using DMSO as a negative control. Subsequently, the cytoxicity of the compounds was determined in a cell viability assay. The percentage was set to 100% for DMSO-treated cells. Bars represent mean of three samples ± SD. Significant differences compared to wt virus are indicated as follows: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. nd = not detectable.

Figure 2

RNA replication of CVB3 3A mutants in the presence of enviroxime, GW5074, and PIK93. (A, B) HeLa cells were infected with wt or mutant CVB3-Rluc for 30 min. Immediately after infection, Guanidine HCl (GuaHCl), enviroxime, GW5074, or PIK93 were added to the cells (B). Cells were lysed at different time points after infection to determine the intracellular Renilla luciferase activity.

Figure 3

Enviroxime targets PI4KIIIβ directly. Recombinant PI4KIIIβ (A) or PI4KIIIα (B) was incubated with the substrate phosphatidylinositol in the form of Triton micelles and radioactively labeled ATP. After termination of the enzyme reaction, the radioactive ATP incorporated into the micelles is quantified as a measure of PI4K activity. (C) Data were converted to percentage inhibition with respect to positive (wortmannin) and negative (DMSO) controls and also presented as IC50. n.a. = not applicable.

Figure 4

Enviroxime and GW5074 decrease PI4P levels at the Golgi complex. (A) HeLa cells were transfected with a plasmid encoding the PI4P sensor FAPP1-PH-GFP. The next day, cells were treated for 1 h with the indicated compounds and stained with an antibody against PI4KIIIβ. (B) Quantification of the percentage of Golgi-localized FAPP1-PH-GFP and PI4KIIIβ, which was determined by dividing the fluorescence intensity of the Golgi-area by that of the area of the whole cell. The resulting percentage was set to 100% in untreated cells. Bars represent the mean of 10 cells from three different experiments ± SD. Significant differences compared to untreated cells are indicated as follows: ^***P < 0.001. (C) GBF1 and Arf1 were stained in HeLa cells treated for 1 h with the indicated compounds.

Figure 5

Mutant PI4KIIIβ rescues the effect of enviroxime on CVB3 replication. (A) Replication rescue experiment. BGM cells were transfected with HA-tagged PI4KIIIβ-wt, PI4KIIIβ-Y583M, or as negative controls the kinase-dead PI4KIIIβ-D656A or EGFP. Two days post-transfection, cells were infected with CVB3 or CVB3-Rluc in the presence of the indicated compounds. After lysing the cells at 8 h p.i., the total virus titer or the amount of luciferase activity was determined in the samples. (B, C) HeLa cells were co-transfected with plasmids encoding FAPP1-PH-GFP and either HA-tagged PI4KIIIβ-wt (B) or PI4KIIIβ-Y583M (C). The next day, cells were treated for 1 h with the indicated compounds. Subsequently, cells were fixed and the overexpressed PI4KIIIβ was stained using an antibody against HA. The percentage of Golgi-localized FAPP1-PH-GFP was determined by dividing the fluorescence intensity of the Golgi-area by that of the area of the whole cell. The resulting percentage was set to 100% in untreated cells. Bars represent the mean of 10 cells from three different experiments ± SD. Significant differences compared to untreated cells are indicated as follows: ^**P < 0.01, ^***P < 0.001.

Figure 6

3A-H57Y does not counter the decrease in PI4P levels induced by enviroxime, GW5074, and PIK93. HeLa cells were co-transfected with plasmids encoding FAPP1-PH-GFP and myc-tagged wt 3A (A) or 3A-H57Y (B). One day after transfection, cells were treated for 1 h with compounds. Subsequently, 3A and 3A-H57Y were stained with an anti-myc antibody. The FAPP1-PH-GFP to 3A fluorescence was quantified in 10 cells per condition and expressed as a ratio. Bars represent the mean ± SD. Significant differences compared to untreated cells are indicated as follows: ^***P < 0.001.

Figure 7

Replication of CVB3 3A mutants is largely independent of PI4KIIIβ. (A) HeLa cells were infected with wt CVB3 or mutant CVB3 3A-H57Y. At 5 h p.i., cells were fixed and stained with antibodies against PI4KIIIβ and dsRNA as a marker for the sites of RNA replication. (B, C) HeLa cells were transfected with siRNAs against PI4KIIIβ (B), or PI4KIIα, PI4KIIβ, and PI4KIIIα (C) and scrambled siRNA as a negative control. Two days later, siRNA-treated cells were infected with wt virus, 3A-V45A virus, or 3A-H57Y virus. Virus titers were determined at 8 h p.i. Bars represent mean of three samples ± SD, ^*P < 0.05. (D) In parallel, efficiency of the knock-down was determined by western blot analysis.

Figure 8

CVB3 3A mutants do not require a high level of PI4P lipids. HeLa cells were infected with CVB3 wt or CVB3 3A-H57Y, after which the medium was replaced with (compound-containing) medium. At 5 h p.i., cells were fixed and stained with antibodies against 3A and PI4P. The PI4P to 3A fluorescence was quantified in 10 cells per condition and expressed as a ratio. Bars represent the mean ± SD. Significant differences compared to untreated cells are indicated as follows: ^***P < 0.001.