Shepard D A, Heinz B A, Rueckert R R
Institute for Molecular Virology, University of Wisconsin, Madison 53706-1596.
J Virol. 1993 Apr;67(4):2245-54. doi: 10.1128/JVI.67.4.2245-2254.1993.
WIN compounds inhibit attachment of human rhinovirus 14 by binding to a hydrophobic pocket within the capsid and inducing conformational changes in the canyon floor, the region that binds the cellular receptor. To study the basis of drug resistance, we isolated and characterized a family of human rhinovirus 14 mutants resistant to WIN 52035-2. Thermostabilization data and single-cycle growth curves provided evidence for two classes of resistant mutants. One class, here called exclusion mutants, showed a marked decrease in drug-binding affinity and was characterized by substitution to bulkier amino acid side chains at two sites lining the hydrophobic pocket. The other class, called compensation mutants, displayed single-amino-acid substitutions in the drug-deformable regions of the canyon; these mutants were able to attach to cells despite the presence of bound drug. A delay in the rise period of the growth curves of compensation mutants indicated a second locus of drug action. WIN 52035-2 was found to inhibit the first step of uncoating, release of VP4. Attempts to identify this site of drug action by using single-step growth curves were obscured by abortive elution of a major fraction of cell-attached virus. The drug had no effect on the rate of this process but did affect the spectrum of particles produced.
WIN化合物通过与衣壳内的疏水口袋结合并诱导峡谷底部（即与细胞受体结合的区域）的构象变化，来抑制人鼻病毒14的附着。为了研究耐药性的基础，我们分离并鉴定了一组对WIN 52035-2耐药的人鼻病毒14突变体。热稳定性数据和单循环生长曲线为两类耐药突变体提供了证据。一类，这里称为排斥突变体，显示出药物结合亲和力显著降低，其特征是在疏水口袋内衬的两个位点被替换为更大的氨基酸侧链。另一类，称为补偿突变体，在峡谷的药物可变形区域显示单氨基酸替换；尽管存在结合的药物，这些突变体仍能够附着于细胞。补偿突变体生长曲线上升期的延迟表明了药物作用的第二个位点。发现WIN 52035-2抑制脱壳的第一步，即VP4的释放。通过单步生长曲线确定该药物作用位点的尝试因大部分细胞附着病毒的流产洗脱而变得模糊不清。该药物对这一过程的速率没有影响，但确实影响了产生的颗粒谱。
