Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

PLoS Pathogens

Bart L. Haagmans¹  Sander I. van Kasteren²  Lucas Pelkmans³  Christine Burkard⁴  Berend Jan Bosch⁴  Frank J. van Kuppeveld⁴  Peter J. M. Rottier⁴  Cornelis A. M. de Haan⁴  Monique H. Verheije⁴  Oliver Wicht⁴ 

[1] Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands;Division of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands;Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland;Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

关键词: Cell fusion;    Lysosomes;    Small interfering RNAs;    Endocytosis;    Endosomes;    Coronaviruses;    Proteases;    Virions;   

DOI  :  10.1371/journal.ppat.1004502

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY