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

When we focused on bat-derived cell lines, VSV-G, NiV-F/G and WSN-HA/NA again permitted pseudotype entry without prior trypsin treatment, while pseudotypes harboring 1918-HA/NA or H2N2-HA/NA were only able to transduce some of the bat cell lines after incubation with trypsin ( Fig 2B) . CpKd cells remained refractory to entry mediated by 1918-HA/NA or H2N2-HA/NA but also showed the lowest susceptibility to all other tested pseudotypes. Notably, three bat cell lines (EidNi/41, HypNi/1.1 and EpoNi/22.1) were susceptible to entry of pseudotypes bearing HAL and NAL of batFLUAV (Fig 2B) , demonstrating that surface glycoproteins of batFLUAV can mediate cellular entry. Entry into the three bat cell lines was robust (1-3 log units above the threshold) and required prior treatment of pseudotypes with trypsin, which presumably resulted in the proteolytic activation of HAL. Furthermore, pseudotypes bearing HAL17/NAL10 or HAL18/NAL10 were both able to enter HypNi/ 1.1 and EpoNi/22.1 cells while EidNi/41 cells could only be transduced by pseudotypes bearing HAL18/NAL10 (Fig 2B) , suggesting that batFLUAV of the HL17NL10 and HL18NL11 subtype might exhibit subtle differences in entry efficiency or cell tropism. Finally, HAL-proteins with a C-terminal FLAG tag facilitated host cell entry, although with somewhat reduced efficiency as compared to their untagged counterparts, indicating that the proteins used to study HAL expression and virion incorporation (Fig 1) were functional (data not shown). Taken together, we showed that batFLUAV surface proteins can mediate entry into certain bat cell lines. For further studies on the entry process, we focused on EpoNi/22.1 cells since they showed the highest susceptibility to entry driven by batFLUAV surface proteins.