The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

The following cell lines were used as targets for transduction experiments and were maintained in Dulbecco's modified Eagle's medium (PAA Laboratories), supplemented with 10% fetal bovine serum (Biochrom) and antibiotics: HEK-293T (human embryonic kidney; ATCC CRL-3216), Vero (African green monkey, kidney; ATCC CCL-81) as well as chiropteran cell lines from four different species of fruit bats (Table 1) , RoNi/7 (Rousettus aegyptiacus, kidney), HypNi/1.1 (Hypsignathus monstrosus, kidney) EidNi/41 (Eidolon helvum, kidney) and EpoNi/ 22.1 (Epomops buettikoferi, kidney). A baby hamster kidney cell line (BHK-21; DSMZ No. ACC-61) was solely used for the production of rhabdoviral pseudotypes and was maintained as described for the other cell lines. All cell lines were obtained from collaborators. The fruit bat cell lines were a kind gift of C. Drosten and M. A. Müller (University of Bonn Medical Centre, Bonn/Germany) and were previously described [39] [40] [41] [42] [43] . All cell lines were grown in a humidified atmosphere at 37°C and 5% CO 2 . For passaging and seeding, cells were detached by either resuspension in fresh culture medium (HEK-293T cells) or the use of trypsin/EDTA (PAA Laboratories; Vero, BHK-21, and bat cells [26, 27, [44] [45] [46] [47] by polymerase chain reaction (PCR) and inserted into the pCAGGS vector [48] by conventional cloning strategies. Details on the cloning strategy (e.g. restriction sites and primer sequences) are available upon request. Expression plasmids (pCAGGS-based) for Marburg virus (MARV, strain Musoke; DQ217792.1) and Lloviu virus (LLOV; JF828358.1) were kindly provided by S. Becker (Philipps-University Marburg, Marburg/Germany) and A. Takada (Hokkaido University Research Center for Zoonosis Control, Sapporo/Japan), respectively [49, 50] . In addition, mutant EBOV-GP in which the furin cleavage motif has been destroyed (EBOV-GP(ΔCleav)) or most of the amino acid (aa) residues comprising the mucin-like domain (MLD, aa residues 309-486) have been deleted (EBOV-GP(ΔMLD)) were constructed by overlap extension PCR from the plasmid containing the wildtype (wt) EBOV-GP sequence. The expression plasmid for the glycoprotein (G) of vesicular stomatitis virus (VSV, Indiana strain, VSV-G; AJ318514.1) was generated by inserting the VSV-G ORF into the pCG1 expression vector and has been used in previous studies [39, 42, 51, 52] .