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

Abstract

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.

Tight Junction Protein Occludin Is a Porcine Epidemic Diarrhea Virus Entry Factor

“…Previous data showed that binding of coronaviruses to target cells is followed by internalization of the viral particles via endocytosis (30). Macropinocytosis is a form of endocytosis which has been identified as an entry mechanism for several viral patho- Results are representative of three independent experiments (means Ϯ SD).…”

Broad-range inhibition of enterovirus replication by OSW-1, a natural compound targeting OSBP

“…Therefore, we tested the effect of OSW-1 on the replication of two other viruses, murine hepatitis virus (MHV), a (+)RNA virus from the Coronaviridae family, and vesicular stomatitis virus (VSV), a (À)RNA virus from the Rhabdoviridae family. Replication of the firefly luciferase-expressing reporter viruses MHV-EFLM (de Haan et al, 2003) or VSVDG/FLuc-G ⁄ (Burkard et al, 2014) was not inhibited by OSW-1, while RLuc-CVB3 replication was significantly inhibited (Fig. 2I).…”

“…and a cell viability was performed in parallel (H). (I) HeLamCC1a cells (Burkard et al, 2014) were infected with RLuc-CVB3, or the MHV-EFLM or VSVDG/FLuc-G ⁄ at an MOI of 1, treated with DMSO, 10 nM OSW-1 or 5 lg/mL BFA as a positive control, cells were lysed at 6 h p.i. and luciferase levels were determined using the (Renilla) Luciferase Assay System kits (Promega).…”

Coronavirus Host Range Expansion and Middle East Respiratory Syndrome Coronavirus Emergence: Biochemical Mechanisms and Evolutionary Perspectives

“…Similarly, the MERS-CoV spike can be processed by TMPRSS2 at the cell surface and by cathepsin L in the endosome (75,76). Furin can also cleave the MERS-CoV spike protein, with increased furin expression resulting in enhanced susceptibility to MERS-CoV (77,78). This finding has important host range implications based on the presence or absence of these proteases in putative host species and the potential differences in cleavage site recognition that might occur between them.…”