The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

Hoffmann, Markus; Krüger, Nadine; Zmora, Pawel; Wrensch, Florian; Herrler, Georg; Pöhlmann, Stefan

Shuttle vectors harboring codon-optimized (for expression in human cells) open reading frames coding for the published amino acid (aa) sequences of the HAL of the two batFLUAV, A/little yellow-shouldered bat/Guatemala/153/2009 (H17/N10) (GenBank: CY103876.1, HAL17) and A/flat-faced bat/Peru/033/2010 (H18/N11) (GenBank: CY125945.1, HAL18), were purchased from a commercial service (Eurofins MWG Operon) and cloned into the pCG1 expression vector, that was kindly provided by R. Cattaneo, via BamHI and XbaI restriction sites. The pCAGGS-based expression plasmid for NAL of batFLUAV A/little yellowshouldered bat/Guatemala/153/2009 (H17/N10) (GenBank: CY103878.1, NAL10) was provided by M. Schwemmle. HAL equipped with a C-terminal FLAG epitope (DYKDDDDK, HAL17-FLAG and HAL18-FLAG) were constructed by PCR and controlled for sequence integrity by automated sequence analysis. In addition, we used pCAGGS-based expression plasmids for the HA and NA of A/WSN/33 (H1N1) and A/Singapore/1/57 (H2N2) (GenBank: AY209895.1) [32, 33] . Furthermore, we employed pCAGGS-based expression plasmids for the HA of A/South Carolina/1/18 (H1N1) (GenBank: AF117241.1) and the NA of A/Brevig Mission/1/1918 (H1N1) (GenBank: AF250356.2) that were generated from previously used constructs [32] . The expression plasmid for the glycoprotein (G) of vesicular stomatitis virus (VSV, Indiana strain, VSV-G; GenBank: AJ318514.1) was generated by inserting the VSV-G ORF into the pCG1 expression vector and has been used in previous studies [28, 31, 34, 35] . Furthermore, expression plasmids for Nipah virus fusion protein (F, GenBank: AF212302) and glycoprotein (G, synthetic, GenBank: AF212302; derived from NiV/MY/HU/1999/CDC) were used [36] . For experiments analyzing proteolytic activation of HAL17 and HAL18 by human type-II transmembrane serine proteases (TTSPs), we employed expression plasmids for TMPRSS2, TMPRSS11E (DESC-1) and TMPRSS13 (MSPL), which have been described previously [32, 33] .