Hemagglutinins of Avian Influenza Viruses Are Proteolytically Activated by TMPRSS2 in Human and Murine Airway Cells

Abstract

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7 and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage site can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analysed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14 and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15 and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage site. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with monobasic cleavage site in ducks. Importance Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and thus represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

Hemagglutinin Subtype Specificity and Mechanisms of Highly Pathogenic Avian Influenza Virus Genesis

“…Many proteases have been identified as likely candidates to cleave monobasic HA0 (reviewed in [ 131 ]). The Type II Transmembrane Serine Protease (TMPRSS2) has been shown to activate most, but not all, human and avian HAs in mice [ 133 , 134 , 135 , 136 , 137 ], human respiratory cell culture models [ 138 , 139 ], and overexpression systems [ 132 ]. Human Airway Trypsin (HAT) is expressed in respiratory ciliated cells and can cleave LPAIV HA0, albeit to a lesser extent than TMPRSS2 [ 132 ].…”

“…Gotoh et al found that a blood clotting factor Xa-like protease from embryonated chicken eggs activates LPAIV HA [ 148 ]. Recently, it has been shown that mallard duck TMPRSS2 can cleave H1–12, H15, and H16, but not the H14 prototype that was used in the study [ 139 ]. The NA of a few specific viral strains can also mediate the non-canonical cleavage of HA0.…”

The Transmembrane Protease TMPRSS2 as a Therapeutic Target for COVID-19 Treatment

“…In order to acquire fusion competence, HA depends on proteolytic cleavage of a linker at the HA1-HA2 interface, a process that triggers conformational rearrangements and enables pH dependent exhibition of the viral fusion peptide [41]. TMPRSS2-mediated cleavage has been described mainly for influenza A viruses; in accordance with the substrate preference of the proteases, cleavage occurs preferentially after a monobasic motif in the linker region [42]. TMPRSS2 was identified as an HA-activating protease in 2006 when Böttcher and colleagues demonstrated TMPRSS2and HAT-(human airway trypsin-like protease, TMPRSS11D)dependent activation of human-pathogenic influenza strains H1N1, H2N9 and H3N2 [43].…”

“…This form is incapable of autoproteolytic activation and lacks proteolytic activity. Consequently, treatment with T-ex5 hampered the replication of monobasic influenza virus strains and SARS-CoV-2 S activation in cell lines and primary cells [7,13,42,56]. In an in vitro screening approach, Chen and colleagues identified therapeutic compounds that reduce TMPRSS2 protein expression in the low-to sub-micromolar range.…”

Endomembrane remodeling in SARS-CoV-2 infection

“…The exposed hydrophobic fusion peptide of S2 inserts into the target cell membrane and mediates membrane fusion to release the viral genome into the cytosol for transcription and replication ( Hoffmann et al, 2021b ; Zhang et al, 2020 ). TMPRSS2 plays important roles in the entry of respiratory viruses, such as SARS-CoV-2, SARS-CoV and Avian Influenza Viruses ( Bestle et al, 2021 ; Glowacka et al, 2011 ; Song et al, 2020 ). Inhibition of TMPRSS2 significantly prevents SARS-CoV and SARS-CoV-2 from entering into target cells ( Hoffmann et al, 2021b ; Kawase et al, 2012 ).…”