Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.
Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.
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DOI：
10.1371/journal.pntd.0004799
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发表时间：
2016-07
影响因子：
3.8
通讯作者：
Tischler ND
中科院分区：
文献类型：
--
作者：
Barriga GP;Villalón-Letelier F;Márquez CL;Bignon EA;Acuña R;Ross BH;Monasterio O;Mardones GA;Vidal SE;Tischler ND
汉坦病毒可导致人类出现汉坦病毒肺综合征或肾综合征出血热。为了进入细胞，汉坦病毒将其包膜与宿主细胞膜融合在一起。以前，我们已经证明了GC包膜糖蛋白是一种与II类融合蛋白具有共同特征的病毒融合蛋白。第二类融合蛋白的胞外结构域由三个结构域组成，这些结构域通过茎区域与病毒包膜中的跨膜锚相连。这些融合蛋白可以通过跨域III(DIII)和茎区域的外源融合蛋白片段被抑制。这些片段被认为在融合前向融合后构象转变的过程中与融合蛋白三聚体的核心相互作用。基于我们之前的安第斯汉坦病毒(ANDV)GC的同源模型结构，我们预测并产生了重组DIII和干肽，以测试这些片段是否抑制汉坦病毒的膜融合和细胞进入。重组ANDV DIII是可溶性的，存在二硫键和β-折叠二级结构，支持In Silico模型。使用DIII和干区的C末端部分，当ANDV在内体途径内融合时，ANDV对细胞的感染被阻断高达60%，当ANDV与质膜发生融合时，ANDV对细胞的感染被阻断高达95%。此外，这些片段还损害了ANDV糖蛋白介导的细胞-细胞融合，并交叉抑制了Puumala病毒糖蛋白(PUUV)介导的融合。GC片段通过阻止膜半融合和孔形成来干扰ANDV细胞的进入，将GC保留在非抗性同源三聚体阶段，如DIII和II类融合蛋白的干肽抑制剂所述。总之，我们的结果表明，汉坦病毒GC不仅在结构上，而且在机制上与II类病毒融合蛋白有相似之处，有望有助于开发针对汉坦病毒的新的治疗策略。包膜病毒对细胞的感染涉及病毒和细胞之间的膜融合。这一过程是由病毒融合蛋白介导的，这些融合蛋白至少分为三个结构类别。膜包膜汉坦病毒是一种在世界范围内传播的病原体，可导致人类疾病，死亡率高达50%，但目前尚无治疗药物和预防措施。在这里，我们证明了安第斯汉坦病毒进入靶细胞可以被来自GC融合蛋白的片段阻断，这些片段类似于II类融合蛋白的抑制片段。GC片段直接作用于病毒融合过程，防止其后期阶段。综上所述，我们的数据表明，汉坦病毒GC蛋白不仅在结构上，而且在机制上与II类融合蛋白相似，表明它是从共同的或相关的祖先融合蛋白进化而来的。此外，研究结果还概述了治疗干预的新方法。
Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses. The infection of cells by enveloped viruses involves the fusion of membranes between viruses and cells. This process is mediated by viral fusion proteins that have been grouped into at least three structural classes. Membrane-enveloped hantaviruses are worldwide spread pathogens that can cause human disease with mortality rates reaching up to 50%, however, neither a therapeutic drug nor preventive measures are currently available. Here we show that the entrance of Andes hantavirus into target cells can be blocked by fragments derived from the Gc fusion protein that are analogous to inhibitory fragments of class II fusion proteins. The Gc fragments acted directly over the viral fusion process, preventing its late stages. Together, our data demonstrate that the hantavirus Gc protein shares not only structural, but also mechanistic similarity with class II fusion proteins, suggesting its evolution from a common or related ancestral fusion protein. Furthermore, the results outline novel approaches for therapeutic intervention.