Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

Faculty of Chemistry, University of Bielefeld, Bielefeld, Germany.
Journal of Virology (Impact Factor: 4.44). 01/2012; 86(7):3736-45. DOI: 10.1128/JVI.06628-11
Source: PubMed


Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.

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    • "For F protein detection, we added a FLAG-tag to the C-terminus of GH-M74a F protein because this has been found to not influence the transport of henipaviral F proteins (Popa et al., 2011). Cloning and characterization of tagged GH-M74a and NiV F and G proteins have been described previously (Diederich et al., 2012; Weis et al., 2014). All mutations were introduced with the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent technologies ) or the Q5 Site Directed Mutagenesis Kit (NEB). "
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    • "Proteolytic activation of the fusion protein of the Nipah virus is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion [136]. Recently cathepsins B and L were reported to be required for the cleavage and productive replication of pathogenic Nipah virus, but not Hendra virus [137]. "
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    • "those of paramyxoviruses) which after proteolytic activation also harbor a free stretch of approximately 20 hydrophobic amino acids, termed the fusion peptide. This fusion peptide inserts into the host-cell membrane to initiate fusion [101] [102]. Although data in this regard is lacking, we hypothesize that the N-terminus of C1 might likewise be able to insert into membranes , for instance of neighbouring cells. "
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