PDF(Pubmed)
Abstract：
Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.
摘要：
鼻病毒（RV）引起鼻腔和肺部反复感染，慢性呼吸系统疾病患者的危及生命的情况，儿童哮喘发作的易感性，和巨大的经济成本。RV很难治疗。它们迅速进化出抗性，并且具有遗传多样性。这里,我们提供了对RV耐药机制对化学化合物中和低pH在内溶酶体。RV-A16在液泡质子ATPase抑制剂巴弗洛霉素A1（BafA1）或内溶体促生剂氯化铵（NH4Cl）存在下的连续传代促进了抗性病毒种群的出现。我们在病毒蛋白1和3（VP1和VP3）中发现了两个可重复的点突变，A2526G（丝氨酸66至天冬酰胺[S66N]），和G2274U（半胱氨酸220至苯丙氨酸[C220F]），分别。两种突变都赋予了对BafA1，NH4Cl的交叉抗性，和质子载体氯硝柳胺，通过大规模平行测序和反向遗传学确定，但不是双重突变，我们无法拯救。VP1-S66和VP3-C220都位于原粒间表面，它们的突变增加了病毒体对低pH的敏感性，温度升高,和可溶性细胞间粘附分子1受体。这些结果表明RV在低内体pH下脱包衣的能力赋予病毒体对细胞外应激的抗性。数据支持内体酸化抑制剂作为对抗房车的可行策略，特别是如果抑制剂直接应用于气道。重要性鼻病毒（RV）是引起普通感冒的主要病原体。抗RV药物和疫苗不可用，很大程度上是由于RV的快速进化适应导致抗性突变体和超过160种不同RV类型的抗原的巨大多样性。在这项研究中,我们通过利用RV对宿主靶向的小化学化合物中和内体pH产生抗性的能力，深入了解RV的细胞生物学。正常房车脱衣的重要线索。我们表明，在用内溶酶体酸化抑制剂处理的细胞中生长的RV进化出衣壳突变，从而降低了病毒体对升高温度的稳定性，低pH值,并与重组可溶性受体片段一起孵育。这种适应性成本使得适应中性pH的RV突变体不太可能在自然界中变得普遍。数据支持针对呼吸道病毒的宿主导向药物开发的概念，特别是在低风险的获得功能突变。
