Hemagglutinin Cleavability, Acid Stability, and Temperature Dependence Optimize Influenza B Virus for Replication in Human Airways

Abstract

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population. IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.

Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

“…As described above, murine hepsin and prostasin were able to activate H3 and IBV HA. Interestingly, human prostasin and hepsin have been shown not to be able to activate H3 in previous studies by us and others (10,11,31). Here, we confirmed these observations.…”

“…7A). A minor band for HA1 was detected upon co-expression of H3 and human hepsin as described before (11). Moreover, no spread of Aichi/H3N2/PR8 was observed in MDCK cells transiently expressing human prostasin or hepsin (Fig.…”

“…Murine factor X, granzyme A, KLK14 and neurotrypsin have not been tested for HA cleavage so far. Previous studies using human proteases showed that factor Xa (activated form of factor X) does not cleave IAV HA with monobasic cleavage site including H3 and that KLK14 is not able to activate IBV HA in vitro (11,37).…”

“…In 2006, we identified the type II transmembrane serine proteases (TTSP) transmembrane serine protease 2 (TMPRSS2) and human airway trypsin-like protease (HAT, also designated as TMPRSS11D) as first human proteases activating IAV HA with monobasic cleavage site in vitro (6). Thereafter, a number of human TTSPs have been shown to activate IAV HA and more recently IBV HA with monobasic cleavage site in vitro (7)(8)(9)(10)(11). In addition, human kallikrein (KLK) 1 (also known as tissue kallikrein) and the kallikrein-related peptidases KLK5 and KLK12 were shown to cleave IAV HA, but not IBV HA with monobasic cleavage site in vitro (11)(12)(13).…”

“…Thereafter, a number of human TTSPs have been shown to activate IAV HA and more recently IBV HA with monobasic cleavage site in vitro (7)(8)(9)(10)(11). In addition, human kallikrein (KLK) 1 (also known as tissue kallikrein) and the kallikrein-related peptidases KLK5 and KLK12 were shown to cleave IAV HA, but not IBV HA with monobasic cleavage site in vitro (11)(12)(13). Further studies demonstrated that TMPRSS2 (also designated as epitheliasin in mice) is essential for activation and spread, and consequently pathogenesis of H1N1pdm, H7N9 and H10 IAV in mice (14)(15)(16)(17)(18).…”