A Mutant H3N2 Influenza Virus Uses an Alternative Activation Mechanism in TMPRSS2 Knockout Mice by Loss of an Oligosaccharide in the Hemagglutinin Stalk Region

Abstract

e The host protease TMPRSS2 plays an essential role in proteolytic activation of the influenza A virus (IAV) hemagglutinin (HA) protein possessing a monobasic cleavage site. However, after passages in TMPRSS2 knockout mice, an H3N2 subtype IAV began to undergo cleavage activation of HA, showing high virulence in the mice due to the loss of an oligosaccharide at position 8 in the HA stalk region. Thus, the H3N2 IAV acquired cleavability by an alternative HA activation mechanism/protease(s). Influenza A virus (IAV), a member of the Orthomyxoviridae family, affects and kills many humans worldwide. The viral hemagglutinin (HA) glycoprotein, which is responsible for receptor binding and subsequent membrane fusion, is synthesized as the inactive precursor, HA 0 , and cleaved by a host cell protease(s) into HA 1 and HA 2 subunits. The cleavage is essential for HA to mediate membrane fusion.Using mouse models, three research groups recently demonstrated that a type II transmembrane serine protease, TMPRSS2, expressed in the airway epithelium, is critically important for HA cleavage in vivo (1-3). Mice lacking TMPRSS2 expression (TMPRSS2 knockout [tmprss2 KO ] mice) are highly tolerant of challenge infection by low-pathogenic (LP) IAVs possessing a monobasic cleavage site in HA. Our group (1) and Hatesuer et al.(2) showed that TMPRSS2 is essential for proteolytic activation of both H1N1 and H3N2 subtype IAVs. However, Tarnow et al. (3) reported that TMPRSS2 is not critical for activation of H3N2 IAV. Since the three groups used different H3N2 IAV strains, the discrepancy likely arose from possible variations in HA among the H3N2 IAV strains. The present study reveals a molecular determinant that modulates the TMPRSS2 dependency of H3N2 IAV in mouse lungs.A mouse-adapted human H3N2 IAV strain, MA-A/Guizhou-X (H3 WT ) (1), was used as a reference wild-type (WT) virus. The virus was a reassortant H3N2 virus of A/Guizhou/54/89 (H3N2), a Chinese human isolate from 1989, and A/Puerto Rico/8/34 (H1N1) (4). The H3WT virus was passaged 10 times in tmprss2 KO mice, and a tmprss2 KO mouse-adapted MA-A/Guizhou-X virus ( H3 p10 ) was obtained.The parental H3 WT and H3 p10 viruses were inoculated intranasally into tmprss2 KO mice (n ϭ 5). After challenge infection with 3.0 ϫ 10 3 PFU of H3 WT , tmprss2 KO mice showed no body weight loss, while all TMPRSS2 ϩ/ϩ wild-type mice (tmprss2 WT ) showed severe body weight loss (Fig. 1A). Conversely, both tmprss2 KO and tmprss2 WT mice infected with 1.7 ϫ 10 2 PFU of H3 p10 showed severe body weight loss and required euthanasia on day 6 postinfection (p.i.) (Fig. 1B). To compare the susceptibility between the two mouse strains in detail, both mouse strains were also infected with 1.7 ϫ 10, 1.7, and 0.17 PFU of H3 p10 ( Fig. 1C to E). The H3 p10 infections exhibited pathogenicity in both tmprss2 KO and tmprss2 WT mice, although the pathological changes were slightly less severe in tmprss2 KO mice than in tmprss2 WT mice. The 50% lethal doses of H3 p10 for tmprss2 WT and tmprss2 KO mice w...

Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice

“…A third study described an H3N2 variant that was not able to replicate in Tmprss2 À/À mice [11]. However, after serial passages in Tmprss2 À/À mice, this virus acquired a mutation in the HA stalk which led to replication and pathology in Tmprss2 À/À mice [13]. Moreover, we recently demonstrated that in Tmprss2 À/À Tmprss4 À/À double knock-out mice, replication and pathology after infection with our H3N2 isolate was strongly diminished and infected mice survived.…”

“…In resistant Tmprss2 À/À mice, H1-and H7-HA are not efficiently cleaved and subsequently the virus fails to spread in infected lungs and therefore does not cause pathology [10][11][12]. On the other hand, H3N2 is able to replicate in Tmprss2 À/À mice and cause mortality [10,13]. In Tmprss2 À/À Tmprss4 À/À double KO mice, replication of H3N2 is still occurring although at a strongly reduced level resulting in low mortality [14].…”

Airway proteases: an emerging drug target for influenza and other respiratory virus infections

“…Compared to WT, TMPRSS2-KO mice proved equally [15] or slightly less vulnerable [14 ] to lethal H3N2 infection. A third H3N2 strain was avirulent in TMPRSS2-KO mice but became lethal after ten passages in mice [16,18]. Since the passaged virus carried an N-glycosylation mutation at the bottom of the HA stalk region, the loss of this glycan may alter the accessibility of the cleavage loop and provide access to an alternative host protease [18].…”

TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1

“…Moreover, polymorphisms in the TMPRSS2 promoter were found to be associated with the severity of influenza in humans (14). Finally, analysis of Tmprss2 knockout mice revealed that Tmprss2 is essential for spread and pathogenesis of several influenza A virus (FLUAV) subtypes (15)(16)(17), although certain H3N2 viruses seem to be able to use proteases other than TMPRSS2 for HA activation (18,19). HAT, DESC1, TMPRSS4, and other TTSP members can activate HA upon directed expression in cell culture (9, 10, 20 -23), and Tmprss4 was shown to contribute to H3N2 FLUAV spread in mice (18).…”