Abstract
Endocytosis plays a particular role in the proteolytic activation of highly pathogenic henipaviruses Hendra (HeV) and Nipah virus (NiV) fusion (F) protein precursors. These proteins require endocytic uptake from the cell surface to be cleaved by cellular proteases within the endosomal compartment, followed by recycling to the plasma membrane for incorporation into budding virions or mediation of cell-cell fusion. This internalization largely depends on a tyrosine-based consensus motif for endocytosis present in the cytoplasmic tail of HeV and NiV F. Given the large number of tyrosine residues present in the F protein cytoplasmic domain of Cedar virus (CedV), a closely related but low pathogenic henipavirus, we aimed to investigate whether CedV F protein undergoes signal-mediated endocytosis from the cell surface controlled by tyrosine-based motifs present in its cytoplasmic tail and whether endocytosis is relevant for its biological activity. Therefore, tyrosine-based signals were mutated, and mutations were assessed for their effect on F cell surface expression, endocytosis, and biological activity. A membrane-proximal YXXΦ motif and a C-terminal di-tyrosine motif are of particular importance for cell surface expression and endocytosis rate. Furthermore, our data strongly indicate the pivotal role of endocytosis for the biological activity of the CedV F protein.
Highlights
Cedar virus (CedV) belongs to the Henipavirus genus within the Paramyxoviridae family and was first isolated from bat urine samples collected from an Australian Pteropus colony in 2012 [1]
In order to investigate whether CedV F protein undergoes endocytosis from the cell surface, we performed a qualitative antibody uptake assay as described previously [11]
Irrespective of the F protein combination, the G expression at the cell surface seems to be similar suggesting that observed effects in fusion activity are not related to differences in CedV G cell surface expression. These findings demonstrate that a membrane-proximal YXXΦ, as well as a C-terminal di-tyrosine motif in the CedV F protein cytoplasmic tail, are of functional relevance for endocytosis and biological activity of the protein
Summary
Cedar virus (CedV) belongs to the Henipavirus genus within the Paramyxoviridae family and was first isolated from bat urine samples collected from an Australian Pteropus colony in 2012 [1]. Despite its genetic proximity to the highly pathogenic Hendra (HeV) and Nipah viruses (NiV), CedV has caused only asymptomatic infections in small animal models so far [1,2]. Further differences are the receptor usage of the attachment proteins. While highly pathogenic HeV and NiV are known to utilize ephrin-B2, expressed i.e., in endothelial cells and lung tissue, and ephrin-B3, mainly found in the central nervous system, for cell entry [4,5,6,7], CedV is unable to use ephrin-B3 but rather binds to ephrin-B1, which is expressed in different tissues such as salivary glands, esophagus, and lung [8]. Recent studies have considered the distinct receptor usage of the CedV attachment protein to contribute to its reduced pathogenicity [8,9]
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