**1. Introduction**

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]. Therefore, research has focused on unraveling the molecular mechanisms leading to differences in the pathogenicity of these closely related viruses. One of the particularities of CedV is an impaired ability of the immunomodulatory phosphoprotein P to counteract the interferon response in cell culture [1,3]. Further differences are the receptor usage of the attachment proteins. The generally abundant expression of ephrins as cell entry receptors in numerous tissues and the high conservation among species results in a wide variety of susceptible hosts and a broad cell type tropism, which is fundamental to the zoonotic character and the pathogenesis of henipaviruses. 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 [4–7], 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]. Therefore, recent studies have considered the distinct receptor usage of the CedV attachment protein to contribute to its reduced pathogenicity [8,9].

Besides receptor binding, the interaction of the attachment protein G with the viral fusion protein F is a prerequisite for virus entry into the host cell and virus spread. An indispensable step for the biological activity of fusion proteins and thus, viral infectivity, is the proteolytic cleavage of the precursor protein F0 into the two subunits F1 and F2 [10]. Interestingly, proteolytic activation of HeV and NiV F protein differs considerably from that of other paramyxoviruses in terms of subcellular localization and protease usage. After transport along the secretory pathway, newly synthesized HeV and NiV F protein precursors require endocytosis from the cell surface to encounter the activating host cell protease and then become biologically active. Cleavage within the endosomal compartment is then followed by recycling to the cell surface before the incorporation of mature fusogenic F1+F2 heterodimers into newly budding virions [11–17]. Overall, both viral envelope proteins are important determinants of pathogenicity that need to act in concert to promote virus-cell membrane fusion needed for virus entry as well as cell-cell fusion resulting in syncytia formation and thus, virus spread.

While trafficking through early and late endosomes prior to fusion with cellular membranes plays a critical role in virus entry of many viruses such as influenza virus [18,19], ebolaviruses [20,21], and flaviviruses [22,23], it is dispensable during NiV entry [16]. Moreover, other viruses and their glycoproteins hijack endosomal pathways in order to support their replication in infected cells [24,25]. The viral envelope glycoprotein of human immunodeficiency virus 1 (HIV-1) for instance undergoes endocytosis during the viral replication cycle, which is hypothesized to serve as a mechanism to evade the host immune response by reducing its cell surface expression (reviewed in [26,27]). In addition, trafficking of the HIV-1 envelope glycoprotein through the endocytic recycling compartment has been recently described as an essential step for incorporation into virus particles [28]. Interestingly, endocytosis of herpesvirus glycoproteins has been discussed to play a functional role in cell–cell fusion and in the production of infectious particles by delivering the glycoproteins to the intracellular site where virus assembly takes place [25,29,30]. Noteworthy, a recent report even suggests that endocytic trafficking of HeV F protein rather than its proteolytic cleavage is a crucial step for efficient HeV virus-like particle (VLP) assembly [31].

Apart from its importance for the viral replication cycle, endocytosis represents a key process for numerous cellular functions. Characterized by the internalization of the plasma membrane and extracellular molecules from the cell surface into internal membrane compartments, endocytosis is required for many biological events such as maintaining the plasma membrane composition or transporting selected cargo molecules from the cell surface to the interior [32]. Among the different types, clathrin-mediated endocytosis (CME) is the best characterized [33,34]. CME of a transmembrane protein is a highly coordinated process that primarily involves the interaction between the cytoplasmic domain of the protein and intracellular adaptor proteins (AP) that select transmembrane cargo into clathrin-coated pits via association to the clathrin lattice. These adaptor complexes recognize specific sorting motifs within the cytoplasmic tail of the cargo protein that are usually tyrosine- or leucine-based [35–38]. One typical sorting motif in cytoplasmic tails of transmembrane proteins known to interact with specific cytosolic adaptor complex proteins is the tyrosine-based YXXΦ motif, in which X can be any amino acid and Φ stands for an amino acid with a large hydrophobic residue. Numerous studies describe YXXΦ motifs to be recognized by AP2 that selects cargo from the plasma membrane on the one hand and facilitates binding to clathrin on the other hand culminating in CME of selected transmembrane cargo [35–41]. However, adaptor complex proteins are not only known to mediate endocytic uptake from the cell surface. They have also been reported to be involved in distinct transport pathways e.g., from early endosomes to recycling or late endosomes, as well as to lysosomes by adapting to specific motifs [38,42]. Clathrin-mediated endocytic uptake of HeV and NiV F proteins strongly depends on a characteristic YXXΦ motif located in the membrane-proximal part of the cytoplasmic tail as depicted in Table 1 [11,12,43]. Consequently, disruption of this motif led to measurable effects in the biological activity of the proteins [11,12] while another di-tyrosine motif in the C-terminal part of the tail had only negligible effects on NiV F endocytosis [11].

**Table 1.** Boldface and underlined letters highlight (potential) endocytosis signals. Numbers above the sequence indicate the amino acid position. Cytoplasmic tails for NiV and HeV F proteins range from amino acid positions 519 to 546, cytoplasmic tail for CedV F is predicted for amino acid positions 517 to 557. Basic aa's that have been shown to be of importance for fusion protein functionality are highlighted in orange.


The cytoplasmic tail of CedV F protein displays several tyrosine residues that might act as functional endocytosis motifs. In addition to a C-terminal di-tyrosine motif similarly found in HeV and NiV F protein, a second di-tyrosine motif is present. Interestingly, two degenerate motifs of the Yxx Φ are obvious: A YxxN motif that has been shown to be a functional endocytosis motif for the spike protein of the Porcine Epidemic Diarrhea Virus [44] and a YxxN motif that has been described to function as an endocytosis signal in the Measles virus hemagglutinin [45]. Thus, in this study, we aimed to investigate the impact of those tyrosine-based motifs on cell surface expression, endocytosis, and biological activity of the protein. Our results sugges<sup>t</sup> a signal-mediated uptake of CedV F protein and confirm the functional importance of a membrane-proximal YXX Φ and a C-terminal di-tyrosine motif for the biological activity of CedV F protein.

### **2. Materials and Methods**

### *2.1. Cell Lines, Transfection*

Vero76, MDCK-2 and HeLa cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loe ffler-Institut, FLI; CCLV-RIE 0228, 1061 and 0082, respectively) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (10%; DMEM10) and incubated at 37 ◦C. Vero76 and MDCK-2 cells were reverse transfected by using the Lipofectamine 3000 reagen<sup>t</sup> (Invitrogen, Schwerte, Germany) following the manufacturer's protocol. HeLa cells were reverse transfected by using the METAFECTENE ® transfection reagen<sup>t</sup> (Biontex, Munich, Germany) according to the manufacturer's instructions.

### *2.2. Plasmids and Site-Directed Mutagenesis*

The open reading frame (ORF) of CedV F and G (GenBank accession no. NC\_025351.1) were synthesized by GeneArt (Thermo Fisher Scientific Inc., Regensburg, Germany) and subcloned into the pCAGGS expression vector. The ORF of the CedV F gene was codon-optimized for expression in human cells and HA-tagged at the C-terminus and will be designated as wild-type (wt) in the manuscript. Selected tyrosine residues within the cytoplasmic domain of CedV F protein were mutated to alanine by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Waldbronn, Germany) and primers designed according to manufacturer's instructions.

### *2.3. Generation of Polyclonal Antibodies*

To obtain a polyclonal antiserum raised against the CedV F protein, a rabbit was immunized subcutaneously three times at three-week intervals with CedV F protein. The antigen was produced in 293T cells as described earlier [46]. Briefly, plasmid DNA encoding for CedV F protein was used to transfect 293T cells. At 48 h post-transfection (p.t.), the supernatant was purified through a 20% sucrose cushion at 96,000× *g* for 2 h at 4 ◦C. After washing for 30 min at 155,000× *g*, pellets were resuspended in Tris-sodium chloride bu ffer. Immunization experiments were assessed and approved

by the competent authority for animal welfare issues of the Federal State of Mecklenburg-Western Pomerania, Germany (license number: LALLF 7221.3-2-042/17).

### *2.4. Antibody Uptake Assay*

MDCK-2 cells were reverse transfected with plasmids encoding for parental and mutant CedV F proteins (1 μg/well) in a 24 well plate as mentioned above. At 24 h p.t., confluent monolayers were washed and incubated without prior fixation with the polyclonal anti-CedV F rabbit serum (1:200 in 0.35% bovine serum albumin (BSA) in PBS++ (PBS with 0.5 mM MgCl, 1 mM CaCl2) (0.35% BSA/PBS++)). After incubation at 4 ◦C for one hour, cells were intensely washed and then either kept on ice or incubated with pre-warmed medium for 30 min at 37 ◦C to allow endocytic uptake. After fixing the cells with 4% paraformaldehyde (PFA), surface-bound primary antibodies were detected by an Alexa-Fluor (AF) 488-labeled secondary antibody (1:50 in 0.35% BSA/PBS++; LifeTechnologies, Darmstadt, Germany) for 90 min at 4 ◦C. After permeabilization with methanol-acetone (1:1) for 10 min, AF 568-labelled secondary antibodies (1:500 in 0.35% BSA/PBS++; LifeTechnologies, Darmstadt, Germany) were added to stain internalized primary antibodies. Images were acquired with an Eclipse Ti-S inverted microscope system and were processed with the ImageJ software version 1.45 s [47].
