Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackievirus B3 (CVB3), substitution N2D in 2C was identified in each of the PI4KB-inhibitor resistant CVB3 pools, but its possible benefit has not been investigated yet. In this study, we set out to investigate the possible role of 2C-N2D in the resistance to PI4KB and OSBP inhibition. We show that 2C-N2D by itself did not confer any resistance to inhibitors of PI4KB and OSBP. However, the double mutant (i.e., 2C-N2D/3A-H57Y) showed better replication than the 3A-H57Y single mutant in the presence of inhibitors. Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the poliovirus polyprotein. Therefore, we studied the effect of PI4KB or OSBP inhibition on proteolytic processing of the CVB3 polyprotein during infection as well as in a replication-independent system. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y recovered impaired proteolytic processing at this junction. Although 2C-N2D enhanced replication of the 3A-H57Y single mutant, we did not detect additional effects of this substitution on polyprotein processing, which leaves the mechanism of how 2C-N2D contributes to the resistance to be revealed.

肠病毒（例如脊髓灰质炎病毒，柯萨奇病毒和鼻病毒）需要几个宿主因子来进行基因组复制。在这些宿主因子中有磷脂酰肌醇-4-激酶IIIβ（PI4KB）和氧固醇结合蛋白（OSBP）。先前已分离出了对PI4KB和OSBP抑制剂具有抗性的肠道病毒突变体，该突变体证明了非结构性3A蛋白中的单取代在赋予抗性中的作用。除了3A替代项（即，在柯萨奇病毒B3（CVB3）中的3A-I54F和3A-H57Y）中，在每个PI4KB抑制剂抗性CVB3库中均确定了2C中的N2D取代，但尚未研究其可能的益处。在这项研究中，我们着手研究2C-N2D在抗PI4KB和OSBP抑制中的可能作用。我们显示2C-N2D本身不赋予PI4KB和OSBP抑制剂任何抗性。但是，双突变体（即，2C-N2D / 3A-H57Y）在抑制剂存在下显示出比3A-H57Y单个突变体更好的复制。越来越多的证据表明，脂质稳态的改变会影响脊髓灰质炎病毒多蛋白的蛋白水解过程。因此，我们研究了PI4KB或OSBP抑制对CVB3多蛋白在感染过程中以及在复制独立系统中的蛋白水解过程的影响。我们显示，PI4KB和OSBP抑制剂都特别影响3A-3B交界处的切割，并且突变3A-H57Y恢复了在此交界处受损的蛋白水解过程。尽管2C-N2D增强了3A-H57Y单个突变体的复制，但我们没有检测到这种取代对多蛋白加工的其他影响，这留下了2C-N2D如何促进抗性的机制。