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

FLUAV-HA is synthesized as an inactive precursor and requires activation by host cell proteases to be responsive to low pH, the trigger for HA-driven membrane fusion [12] [13] [14] . Members of the TTSP family activate FLUAV-HA in cell culture [33, [54] [55] [56] [57] [58] and TMPRSS2 was previously shown to be essential for FLUAV-HA activation and viral spread in mice [59] . Therefore, we asked whether TTSPs able to activate FLUAV-HA can also activate batFLUAV-HAL. For this, we first investigated batFLUAV-HAL cleavage by TMPRSS2, DESC-1 and MSPL, and compared it to cleavage by trypsin. Cleavage of the 1918-HA served as positive control. We found that 1918-HA was efficiently processed by all proteases tested, as expected. Moreover, we could show that coexpression of TMPRSS2, DESC-1 and MSPL, and trypsin treatment resulted in cleavage of the HAL precursor (HAL 0 ) determined by the appearance of bands corresponding to the HAL 2 subunit (Fig 6A) . While HAL18 was comparably cleaved by all tested TTSPs, HAL17 cleavage by TMPRSS2 was more pronounced than proteolysis by DESC-1 and MSPL (Fig 6A) . Moreover, HAL18 was generally more sensitive to cleavage by TTSPs than HAL17 (Fig 6A) . In order to assess whether batFLUAV-HAL cleavage by TTSPs also leads to HAL activation for host cell entry, we produced pseudotypes harboring batFLUAV-HAL (HAL17 or HAL18) in the presence of TMPRSS2, DESC-1 and MSPL. As a control, pseudotypes bearing 1918-HA and -NA were included in this experiment. The pseudotypes were treated with trypsin to activate HA/HAL or were mock-treated before addition to EpoNi/22.1 cells. Pseudotypes bearing 1918-HA and -NA and produced in the presence of TMPRSS2, DESC-1 and MSPL or treated with trypsin robustly transduced target cells ( Fig 6B) . In contrast, infectivity of FLUAV-HA pseudotypes produced in the absence of TTSPs or not treated with trypsin was in the background range (Fig 6B) . Similarly, batFLUAV-HALbearing pseudotypes were activated by trypsin or TTSPs, including TMPRSS2 (Fig 6B) . However, differences in the activation of HAL17 and HAL18 were observed and correlated with the efficiency of HAL protein cleavage, as determined above (Fig 6A) . Thus, expression of TMPRSS2 but not DESC-1 and MSPL conferred robust infectivity to HAL17-bearing pseudotypes while all proteases were able to efficiently activate HAL18. Moreover, transfection of escalating amounts of TMPRSS2-encoding plasmids increased infectivity of HAL17-bearing pseudotypes in a concentration-dependent manner. In contrast, transfection of even the lowest amount of TMPRSS2 plasmid was sufficient to confer maximal infectivity of HAL18bearing pseudotypes, confirming that the efficiency of TMPRSS2-mediated activation of HAL is subtype specific (Fig 6C) . In sum, proteolytic activation of batFLUAV-HAL is critical for HAL-driven cell entry and proteases able to activate HA can also activate HAL. Host Cell Entry by Bat-Associated Influenza Viruses