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

The identification of two new FLUAV, subtypes H17N10 (HL17NL10) and H18N11 (HL18NL11), in new-world bats [16, 17] suggests that bats could serve as a natural reservoir of FLUAV [16, 17] . Unraveling the zoonotic potential of batFLUAV is of great importance since FLUAV are major human pathogens, responsible for influenza epidemics and pandemics. While the batFLUAV replication machinery appears to be functional in different mammalian (including human) cells [16, [18] [19] [20] [21] 60] , reassortment with FLUAV and FLUBV is unlikely [18, 19] . Regarding batFLUAV tropism of the viral surface proteins, HAL and NAL, only Human proteases that activate FLUAV-HA for cell entry also activate batFLUAV-HAL. (A) HEK-293T cells were transfected with plasmids encoding HA or HAL proteins and either trypsin treated or cotransfected with plasmids encoding type II transmembrane serine proteases. Transfection of empty vector served as negative control. Cleavage of HA/HAL proteins was analyzed by SDS-PAGE and Western blotting, employing antibodies against FLUAV-HA (α-FLUAV) and the FLAG epitope (α-FLAG). Detection of ß-actin served as loading control. Signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. The results were confirmed in a separate experiment. To assess proteolytic activation of HA/HAL proteins, vesicular stomatitis virus-based pseudotypes (VSVpp) were produced in cells transfected to express the indicated type II transmembrane serine proteases (B) or different amounts of TMPRSS2 (C). Pseudotypes were either directly used for transduction of EpoNi/22.1 cells (black bars) or previously treated with trypsin (white bars). At 24 h post inoculation, transduction efficiency was measured by quantification of the activity of VSVpp-encoded luciferase in cell lysates. For normalization, transduction by HA-or HAL-bearing pseudotypes that were produced in the absence of type II transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. The result of a single representative experiment carried out with quadruplicate samples is presented. Similar results were obtained in three independent experiments carried out with separate pseudotype preparations. Error bars indicate standard deviations. A two-tailed, unpaired student's t-test was used to test statistical significance (* = p < 0.05).