Bayzid Md, Bhowmick Biswajit, Ahmed Waqas, Neelakanta Girish, Sultana Hameeda
Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
Viruses. 2025 Jul 10;17(7):969. doi: 10.3390/v17070969.
GW4869, a cell-permeable, selective inhibitor of neutral sphingomyelinase is a pharmacological agent that blocks the production and release of extracellular vesicles (EVs). Our previous studies have shown that GW4869 inhibits flaviviral loads in tick, mosquito and mammalian cells, including murine cortical neurons. Yet the mechanism(s) of GW4869 inhibitor upon viral infections were not addressed. In the current study, we focused on how GW4869 interferes with Langat Virus (LGTV, a tick-borne flavivirus) replication in ISE6 tick cells. First, we found that GW4869 is neither cytotoxic at tested doses of 50, 100, and 150 µM in tick cells, nor does it directly bind to the free LGTV present in cell culture supernatants. When tick cells were treated with GW4869, followed by infection with viral stock at dilutions of 10, 10, 10 (the infectious dose determination by viral dilution assay), it affected LGTV replication in tick cells. A reduction in viral burden was noted in GW4869-treated tick cells, which constituted more than half the amount of decrease when compared to the mock control. Next, GW4869 treatment not only resulted in decreased LGTV transcript levels in tick cells and EVs derived from these infected cells, but also revealed diminished EVs concentrations. Enhanced SMase transcripts in the LGTV-infected group was noted upon GW4869 treatment, thus suggesting a host response to perhaps inhibit virus replication. In addition, GW4869 treatment reduced LGTV loads in density gradient EVs fractions, which correlated with decreased EVs concentration in those fractions. These data not only indicate that GW4869 affects LGTV replication, but that it also interferes with EV secretion and release from tick cells. Lastly, we found that GW4869 inhibits LGTV replication in tick cells but does not directly affect the infectivity of LGTV viral particles. Overall, our study suggests that GW4869 is a potential therapeutic inhibitor in controlling tick-borne diseases.
GW4869是一种可穿透细胞的中性鞘磷脂酶选择性抑制剂，是一种能阻断细胞外囊泡（EVs）产生和释放的药物制剂。我们之前的研究表明，GW4869可抑制蜱虫、蚊子和哺乳动物细胞（包括小鼠皮层神经元）中的黄病毒载量。然而，GW4869抑制剂对病毒感染的作用机制尚未得到研究。在本研究中，我们重点关注GW4869如何干扰兰加特病毒（LGTV，一种蜱传黄病毒）在ISE6蜱细胞中的复制。首先，我们发现，在蜱细胞中，50、100和150µM的测试剂量下，GW4869既无细胞毒性，也不直接结合细胞培养上清液中游离的LGTV。当用GW4869处理蜱细胞，随后以10、10、10的稀释度感染病毒原液（通过病毒稀释试验确定感染剂量）时，它影响了LGTV在蜱细胞中的复制。在GW4869处理的蜱细胞中，病毒载量有所降低，与模拟对照相比，减少量超过一半。接下来，GW4869处理不仅导致蜱细胞和源自这些感染细胞的EVs中LGTV转录水平降低，还显示出EVs浓度降低。GW4869处理后，LGTV感染组中鞘磷脂酶（SMase）转录本增强，因此表明宿主可能通过某种反应来抑制病毒复制。此外，GW4869处理降低了密度梯度EVs组分中的LGTV载量，这与这些组分中EVs浓度降低相关。这些数据不仅表明GW4869影响LGTV复制，还表明它干扰了EVs从蜱细胞的分泌和释放。最后，我们发现GW4869抑制蜱细胞中的LGTV复制，但不直接影响LGTV病毒颗粒的感染性。总体而言，我们的研究表明，GW4869是控制蜱传疾病的一种潜在治疗抑制剂。
