Watanabe M, Hirano A, Stenglein S, Nelson J, Thomas G, Wong T C
Department of Microbiology, University of Washington School of Medicine, Seattle 98195, USA.
J Virol. 1995 May;69(5):3206-10. doi: 10.1128/JVI.69.5.3206-3210.1995.
We have identified the major cellular endoprotease that activates the fusion (F) glycoprotein of measles virus (MV) and have engineered a serine protease inhibitor (serpin) to target the endoprotease and inhibit the production of infectious MV. The F-protein precursor of MV was not cleaved efficiently into the mature F protein in human colon carcinoma cells lacking functional furin, indicating that furin is the major enzyme responsible for activation of the MV F protein. A human serpin alpha 1-antitrypsin variant was engineered to specifically inhibit furin. When expressed from a recombinant vaccinia virus in primate cells infected by MV, the engineered serpin (alpha 1-PDX) specifically inhibited furin-catalyzed cleavage of the F-protein precursor without affecting synthesis of other MV proteins. We generated human glioma cells stably expressing alpha 1-PDX. MV infection in these cells did not result in syncytia. The infected cells produced all the MV proteins, but the F-protein precursor remained largely uncleaved. This did not prevent virus assembly. However, the released virions contained inactive F-protein precursor rather than mature F protein, and infectious-virus titers were reduced by 3 to 4 orders of magnitude. These results show that a mature F protein is not required for the assembly of MV but is crucial for virus infectivity. The engineered serpin may offer a novel molecular antiviral approach against MV.
我们已经鉴定出激活麻疹病毒(MV)融合(F)糖蛋白的主要细胞内蛋白酶,并设计了一种丝氨酸蛋白酶抑制剂(丝氨酸蛋白酶抑制剂)来靶向该蛋白酶并抑制传染性MV的产生。在缺乏功能性弗林蛋白酶的人结肠癌细胞中,MV的F蛋白前体不能有效地切割成成熟的F蛋白,这表明弗林蛋白酶是负责激活MV F蛋白的主要酶。对人丝氨酸蛋白酶抑制剂α1-抗胰蛋白酶变体进行了工程改造,以特异性抑制弗林蛋白酶。当从重组痘苗病毒在被MV感染的灵长类细胞中表达时,工程化的丝氨酸蛋白酶抑制剂(α1-PDX)特异性抑制弗林蛋白酶催化的F蛋白前体的切割,而不影响其他MV蛋白的合成。我们生成了稳定表达α1-PDX的人胶质瘤细胞。这些细胞中的MV感染未导致多核巨细胞形成。受感染的细胞产生了所有的MV蛋白,但F蛋白前体大部分仍未切割。这并不妨碍病毒组装。然而,释放的病毒粒子含有无活性的F蛋白前体而不是成熟的F蛋白,并且感染性病毒滴度降低了3至4个数量级。这些结果表明,成熟的F蛋白对于MV的组装不是必需的,但对于病毒感染性至关重要。工程化的丝氨酸蛋白酶抑制剂可能提供一种针对MV的新型分子抗病毒方法。