Abstract:
Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC50) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC50 values of 0.31 µM-0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs.IMPORTANCELassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV.
摘要:
拉沙病毒(LASV)是人类拉沙热的病原体,其在严重的情况下表现为每年导致数千人死亡的出血热。然而,目前尚无批准的疫苗或抗病毒药物。最近,我们使用表达LASV糖蛋白GP(VSV-LASVGP)的重组水泡性口炎病毒(VSV)筛选了约2,500种化合物,并鉴定了P-糖蛋白抑制剂为潜在的LASV进入抑制剂.这里,我们显示另一个确定的候选人,hexestrol(HES),雌激素受体激动剂,也是LASV进入抑制剂。HES以0.63μM的50%抑制浓度(IC50)抑制VSV-LASVGP复制。重要的是,HES还抑制真正的LASV复制,IC50值为0.31µM-0.61µM。添加时间和基于细胞的膜融合测定表明HES在病毒进入期间抑制膜融合步骤。替代雌激素受体激动剂不抑制VSV-LASVGP复制,这表明雌激素受体本身不太可能参与HES的抗病毒活性。HES抗性突变体的产生揭示了LASVGP氨基酸位置446(F446)的苯丙氨酸,它位于跨膜区域,赋予了对HES的抵抗力。尽管F446的突变增强了LASVGP的膜融合活性,它表现出减少的VSV-LASVGP复制,很可能是由于LASVGP融合前状态的不稳定性。总的来说,我们的结果表明,HES是一种有前景的抗LASV药物,其通过抑制LASV进入的膜融合步骤起作用.这项研究还强调了LASVGP跨膜区作为抗LASV药物靶标的重要性。IMPORTANCELassa病毒(LASV),拉沙热的病原体,就其对西非公共卫生的影响而言,是最具破坏性的哺乳动物病毒。然而,目前尚无批准的抗病毒药物或疫苗。这里,我们确定了hexestrol(HES),雌激素受体激动剂,作为潜在的抗病毒候选药物。我们表明,雌激素受体本身不参与抗病毒活性。HES直接与LASVGP结合并阻断膜融合,从而抑制LASV感染。通过产生抗HES病毒,我们发现LASVGP跨膜区446位(F446)的苯丙氨酸在HES的抗病毒活性中起着至关重要的作用。F446突变导致病毒复制减少,可能是由于LASVGP融合前状态的不稳定性。这些发现突出了HES作为开发靶向LASV的抗病毒化合物的有希望的候选物的潜力。