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

It has been previously reported that HAL and NAL of batFLUAV are not compatible with viral spread in the cell lines tested so far [18, 19] , suggesting that these proteins mediate entry into a restricted set of target cells or are even inactive. In order to gain insights into the functional activity of batFLUAV-HAL and NAL, we employed rhabdoviral vectors pseudotyped with these proteins. We inoculated cell lines from different host species, including those standardly used for FLUAV research, as well as cell lines derived from different bat species (Table 1) , as they are the natural reservoir for batFLUAV. We chose bat cell lines known to be susceptible to infection by viruses of different families [28, 29, 31] and previously used to functionally characterize surface glycoproteins of bat-borne viruses [28, 36, 38] . It is well established that FLUAV-HA depends on proteolytic cleavage by host cell proteases to transit into an active form [12] and it has been previously reported that batFLUAV can be activated by exogenous trypsin [39] [40] [41] . Therefore, we assessed whether trypsin treatment of the pseudotypes impacts transduction efficiency. As controls, we included the surface glycoprotein(s) of well-characterized FLUAV strains, A/WSN/33 (H1N1) (WSN-HA, WSN-NA), A/South Carolina/1/18 We found that none of the human, simian and canine cell lines was susceptible to entry driven by batFLUAV surface proteins (Fig 2A) . In contrast, pseudotypes bearing VSV-G, NiV-F/G or WSN-HA/NA could readily enter these cells, whereas pseudotypes that harbored 1918-or H2N2-HA/NA required activation by exogenous trypsin for efficient transduction (Fig 2A) . These results are in agreement with expectations, since activation of WSN-HA is known to be independent of trypsin [42] [43] [44] , although viral infectivity can be enhanced by trypsin treatment.