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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Available from: Michael Weis, Oct 02, 2015
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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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    ABSTRACT: Compared to the fusion proteins of pathogenic Nipah and Hendra viruses, the F protein of prototype African henipavirus GH-M74a displays a drastically reduced surface expression and fusion activity. A probable reason for limited F expression is the unusually long sequence located between the gene start and the signal peptide (SP) not present in other henipaviruses. Such a long pre-SP extension can prevent efficient ER translocation or protein maturation and processing. As its truncation can therefore enhance surface expression, the recent identification of a second in-frame start codon directly upstream of the SP in another African henipavirus F gene (GH-UP28) raised the question if such a naturally occurring minor sequence variation can lead to the synthesis of a pre-SP truncated translation product, thereby increasing the production of mature F proteins. To test this, we analyzed surface expression and biological activity of F genes carrying the second SP-proximal start codon of GH-UP28. Though we observed minor differences in the expression levels, introduction of the additional start codon did not result in an increased fusion activity, even if combined with further mutations in the pre-SP region. Thus, limited bioactivity of African henipavirus F protein is maintained even after sequence changes that alter the gene start allowing the production of F proteins without an unusually long pre-SP. Copyright © 2015. Published by Elsevier B.V.
    Virus Research 02/2015; 201. DOI:10.1016/j.virusres.2015.02.016 · 2.32 Impact Factor
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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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    ABSTRACT: Proteinases and their inhibitors play essential functional roles in basic biological processes in both hosts and pathogens. Endo/lysosomal cathepsins participate in immune response in pathogen recognition and elimination. They are essential for both antigen processing and presentation (host adaptive immune response) and activation of endosomal toll like receptors (innate immune response). Pathogens can produce proteases and also natural inhibitors to subvert the host immune response. Several pathogens are sensed through the intracellular pathogen recognition receptors, but only some of them use the host proteolytic system to escape into the cytosol. In this review, I provide an update on the most recent developments regarding the role of proteinases and their inhibitors in the initiation and regulation of immune responses.
    Current Protein and Peptide Science 12/2012; 13(8). DOI:10.2174/138920312804871102 · 3.15 Impact Factor
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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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    ABSTRACT: A variety of physiological functions, not only restricted to the nervous system, are discussed for the cellular prion protein (PrP(C)). A prominent, non-physiological property of PrPC is the conversion into its pathogenic isoform (PrP(Sc)) during fatal, transmissible, and neurodegenerative prion diseases. The prion protein is subject to posttranslational proteolytic processing and these cleavage events have been shown i) to regulate its physiological functions, ii) to produce biologically active fragments, and iii) to potentially influence the course of prion disease. Here, we give an overview on the proteolytic processing under physiological and pathological conditions and critically review what is currently known about the three main cleavage events of the prion protein, namely α-cleavage, β-cleavage, and ectodomain shedding. The biological relevance of resulting fragments as well as controversies regarding candidate proteases, with special emphasis on members of the A-disintegrin-and-metalloproteinase (ADAM) family, will be discussed. In addition, we make suggestions aimed at facilitating clarity and progress in this important research field. The better understanding of this issue will not only answer basic questions in prion biology but will likely impact research on other neurodegenerative diseases as well.
    American Journal of Neurodegenerative Diseases 01/2012; 1(1):15-31.
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