Van der Gucht Winke, Leemans Annelies, De Schryver Marjorie, Heykers Annick, Caljon Guy, Maes Louis, Cos Paul, Delputte Peter L
Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Antwerp, Belgium.
Virol J. 2017 Aug 17;14(1):157. doi: 10.1186/s12985-017-0824-3.
Host proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection.
To measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy.
Several protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3-2 and A2000/3-4, suggesting that this is a general feature of RSV.
RSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.
宿主蛋白酶已被证明在许多病毒活动中发挥重要作用，如病毒进入、脱壳、病毒蛋白产生和疾病诱导。因此，这些细胞蛋白酶是开发抑制其活性的抗病毒药物的潜在靶点。宿主蛋白酶已被描述在埃博拉病毒、丙型肝炎病毒、艾滋病毒和流感中发挥重要作用，因此特定的蛋白酶抑制剂能够减少感染。呼吸道合胞病毒（RSV）在其复制周期中利用宿主蛋白酶，但其作为抗病毒靶点的潜力尚不清楚。因此，我们评估了蛋白酶抑制剂对RSV感染的影响。
为了测量RSV感染对蛋白酶抑制剂的敏感性，将细胞用RSV感染，并在有或没有抑制剂的情况下孵育18小时。细胞固定、染色后用荧光显微镜进行研究。
测试了几种代表不同类别的蛋白酶的蛋白酶抑制剂（AEBSF、胃蛋白酶抑制剂A、E-64、TPCK、PMSF和抑肽酶）对HEp-2细胞RSV A2感染的抑制作用。评估了不同的处理持续时间，从接种前1小时开始并在测定过程中持续18小时。在所有测试的抑制剂中，AEBSF和TPCK显著降低了RSV感染。为了确定观察到的AEBSF的作用不是与HEp-2细胞相关的特定特征，还使用了A549和BEAS-2B细胞。与HEp-2细胞类似，孵育18小时后观察到RSV感染细胞数量几乎完全受阻，且该作用呈剂量依赖性。为了深入了解这种抑制的机制，在感染周期的不同阶段（接种前、接种时和接种后）应用AEBSF处理。这些实验的结果表明，AEBSF主要在RSV的早期进入阶段发挥作用。在其他RSV分离株A1998/3-2和A2000/3-4中也观察到了抑制作用，这表明这是RSV的一个普遍特征。
广泛的丝氨酸蛋白酶抑制剂AEBSF和TPCK可抑制RSV感染。我们证实AEBSF的抑制作用与所用细胞系或RSV毒株无关。发现抑制剂处理最有效的时间点与病毒粒子进入宿主细胞的预期时间一致。
