Ledford Rebecca M, Collett Marc S, Pevear Daniel C
ViroPharma Incorporated, Exton, PA 19341, USA.
Antiviral Res. 2005 Dec;68(3):135-8. doi: 10.1016/j.antiviral.2005.08.003. Epub 2005 Sep 19.
Recent phylogenetic analyses of the deduced amino acid sequence of the major viral capsid protein (VP1) of all human rhinovirus (HRV) serotypes revealed two distinct species within the genus: species A (75 serotypes) and species B (25 serotypes). Pleconaril is a novel capsid inhibitor of HRVs. All 75 species A serotypes and 18 of the 25 species B serotypes are susceptible to inhibition by pleconaril in cell culture. The seven resistant serotypes are HRV-4, -5, -42, -84, -93, -97 and -99. We were interested in understanding the genetic basis for phenotypic resistance to pleconaril among these naturally occurring viruses. We compared the 25 amino acids of VP1 that comprise the drug-binding pocket of susceptible and resistant species B viruses. A consistent difference was observed at two positions: the vast majority of susceptible viruses had tyrosine and valine at VP1 residues 152 and 191, respectively (Y(152) and V(191)); all resistant viruses had phenylalanine and leucine at these positions (F(152) and L(191)). HRV-14, a pleconaril susceptible virus, has a drug-binding pocket amino acid composition that differs from the naturally resistant HRV-5 and HRV-42 only at these two positions. To gain further insight into the role of these specific residues in natural resistance to pleconaril, we substituted the amino acids at these two positions individually and in combination in an infectious clone of HRV-14 and tested the rescued virus for susceptibility to pleconaril and virion stability. The results indicate that substitution of V(191) to Leu in HRV-14 has a profound negative impact on drug susceptibility but that full resistance to pleconaril is only seen when combined with Phe at position 152 in a HRV-14 double variant (F(152), L(191)). These data identify L(191) in species B HRV as a potentially key residue in conferring significantly reduced susceptibility to pleconaril. These results may be useful in distinguishing naturally occurring viral resistance to pleconaril from treatment-emergent resistance.
最近对所有人类鼻病毒（HRV）血清型主要病毒衣壳蛋白（VP1）推导氨基酸序列进行的系统发育分析显示，该属内有两个不同的种：A种（75个血清型）和B种（25个血清型）。普来可那立是一种新型的HRV衣壳抑制剂。在细胞培养中，所有75种A种血清型和25种B种血清型中的18种对普来可那立的抑制敏感。7种耐药血清型为HRV-4、-5、-42、-84、-93、-97和-99。我们感兴趣的是了解这些天然存在的病毒对普来可那立表型耐药的遗传基础。我们比较了构成敏感和耐药B种病毒药物结合口袋的VP1的25个氨基酸。在两个位置观察到一致的差异：绝大多数敏感病毒在VP1残基152和191处分别具有酪氨酸和缬氨酸（Y(152)和V(191)）；所有耐药病毒在这些位置具有苯丙氨酸和亮氨酸（F(152)和L(191)）。HRV-14是一种对普来可那立敏感的病毒，其药物结合口袋氨基酸组成仅在这两个位置与天然耐药的HRV-5和HRV-42不同。为了进一步深入了解这些特定残基在对普来可那立天然耐药中的作用，我们在HRV-14的感染性克隆中分别和组合替换这两个位置的氨基酸，并测试拯救出的病毒对普来可那立的敏感性和病毒体稳定性。结果表明，HRV-14中V(191)替换为亮氨酸对药物敏感性有深远的负面影响，但只有在HRV-14双变体（F(152)，L(191)）中与152位的苯丙氨酸结合时才会出现对普来可那立的完全耐药。这些数据确定B种HRV中的L(191)是赋予对普来可那立敏感性显著降低的潜在关键残基。这些结果可能有助于区分天然存在的对普来可那立的病毒耐药性和治疗中出现的耐药性。
