多种血清型的存在使得疫苗开发对于肠道病毒属中的大多数病毒具有挑战性。控制这些病毒的另一种可能更可行的策略是通过靶向对病毒生命周期必不可少的高度保守的蛋白质来开发广谱抗病毒药物,如3C蛋白酶。以前,两个单链抗体片段,YDF和GGVV,据报道可以有效抑制人鼻病毒14的增殖。这里,我们发现两种单链抗体片段都靶向3C蛋白酶上的位点,这些位点与其已知的药物位点(肽酶活性位点)不同,并且具有不同的抑制机制.YDF不阻断活性位点,而是通过变构效应非竞争性地抑制3C肽酶活性,这在抗体蛋白酶抑制剂中很少见。同时,GGVV拮抗基因组复制中3C的较少探索的调节功能。据报道,3C与病毒基因组5'非编码区之间的相互作用对肠道病毒基因组复制很重要。这里,通过氢-氘交换耦合质谱法探测人鼻病毒143C及其5'非编码区之间的界面,发现其与GGVV和3C之间的界面部分重叠.始终如一,GGVV的预结合完全消除了人鼻病毒143C与其5'非编码区之间的相互作用。YDF和GGVV的表位,因此,代表鼻病毒治疗脆弱性的另外两个位点。重要的是,GGVV表位在许多肠道病毒中似乎是保守的,这表明它是泛肠道病毒抑制剂筛选和设计的一个有希望的靶标。
The existence of multiple serotypes renders vaccine development challenging for most viruses in the Enterovirus genus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5\' noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5\' noncoding region was probed by hydrogen-deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5\' noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design.