Burkard Christine, Verheije Monique H, Wicht Oliver, van Kasteren Sander I, van Kuppeveld Frank J, Haagmans Bart L, Pelkmans Lucas, Rottier Peter J M, Bosch Berend Jan, de Haan Cornelis A M
Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Division of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
PLoS Pathog. 2014 Nov 6;10(11):e1004502. doi: 10.1371/journal.ppat.1004502. eCollection 2014 Nov.
Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.
包膜病毒需要与宿主细胞膜融合，以便将其基因组传递到宿主细胞中。虽然一些病毒与质膜融合，但许多病毒在融合之前会被内吞。内体微环境中的特定信号会诱导病毒融合蛋白发生构象变化，从而导致病毒膜与宿主膜融合。在本研究中，我们调查了冠状病毒（CoV）的进入机制。使用小干扰RNA（siRNA）基因沉默技术，我们发现已知对晚期内体成熟和内体-溶酶体融合很重要的蛋白质能显著促进小鼠肝炎冠状病毒（MHV）对细胞的感染。使用表达报告基因的重组MHV以及一种新型的、与复制无关的融合试验，我们证实了网格蛋白介导的内吞作用的重要性，并证明MHV运输到溶酶体是融合和有效进入所必需的。然而，与分别在早期和晚期内体中融合的水疱性口炎病毒和甲型流感病毒相比，MHV对内体pH值的扰动不太敏感。我们的结果表明，MHV的进入取决于其融合蛋白S被溶酶体蛋白酶进行蛋白水解加工。一种泛溶酶体蛋白酶抑制剂能严重抑制MHV的融合，而当在融合肽上游紧邻的S蛋白中引入弗林蛋白酶切割位点时，就不再需要MHV运输到溶酶体并被溶酶体蛋白酶加工。猫冠状病毒的进入也显示取决于运输到溶酶体并被溶酶体蛋白酶加工。相比之下，中东呼吸综合征冠状病毒（MERS-CoV）在融合肽上游紧邻处含有一个最小的弗林蛋白酶切割位点，它受到弗林蛋白酶抑制的负面影响，但不受溶酶体蛋白酶抑制的影响。我们得出结论，融合肽上游紧邻的CoV S蛋白中的蛋白水解切割位点是细胞内融合位点的一个关键决定因素。
