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.
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Acknowledgements
We are grateful to Drs K Conrath and M Andrews (Galapagos NV, Belgium) for performing the in vitro PI4K activity assay. We also thank Drs N Altan-Bonnet (Rutgers University, USA) and T Balla (National Institute for Health, USA) for fruitful discussions and for providing plasmids, Dr F Wieland (Biochemie-Zentrum, Germany) for providing antibodies and Dr J Albanesi (UT Southwestern Medical Center, Texas) for providing plasmids. This work was supported by research grants from the “Covenant K.U. Leuven-Radboud University Nijmegen” framework, the Netherlands Organization for Scientific Research (NWO-ECHO-700.57.001 and NWO-ALW-820.02.018), and the SILVER Large Scale Collaborative Project (grant agreement number 260644) of the European Union 7th Framework. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Supplementary information, Figure S1
Golgi-localization of FAPP1-PH-GFP is predominantly determined by PI4KIIIβ. (PDF 57 kb)
Supplementary information, Figure S2
Enviroxime, GW5074, and PIK93 do not affect PI4KIIIβ recruitment by wild-type 3A or 3A-H57Y. (PDF 118 kb)
Supplementary information, Figure S3
SiRNA-treatment does not induce cytotoxic effects on cells. (PDF 44 kb)
Supplementary information, Figure S4
Efficient depletion of PI4KIIβ by siRNA-treatment. (PDF 43 kb)
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van der Schaar, H., van der Linden, L., Lanke, K. et al. Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication. Cell Res 22, 1576–1592 (2012). https://doi.org/10.1038/cr.2012.129
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DOI: https://doi.org/10.1038/cr.2012.129
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