The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases

Abstract

Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro. Recently, we reported that inactivation of a single HA-activating protease gene, Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast, Tmprss2−/− Tmprss4−/− double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo.IMPORTANCE Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes, Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza viruses in vivo.

Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs

“…H1N1 virus replication was suppressed in the respiratory tract of TMPRSS2 Ϫ/Ϫ mice, while H3N2 virus replication was only marginally affected (11). It was recently proposed that TMPRSS4 plus TMPRSS2 activate H3N2 viruses in mice (12). We now need to know whether some endogenous kallikreins are involved in the replication and pathogenesis of H3 subtype influenza viruses in mice.…”

“…Single-strand cDNA was synthesized from 1 g total RNA from each sample with the High Capacity cDNA reverse transcription kit (Life Technologies SAS, Saint Aubin, France). For analysis of the viral nucleoprotein mRNA, the reverse transcription step was performed with an oligo(dT) [12][13][14][15][16][17][18] primer (Invitrogen). mRNA levels were determined by quantitative real-time PCR with a LightCycler 480 instrument (Roche Diagnostics, Meylan, France).…”

“…Although TMPRSS2 is a marker of susceptibility to severe A (H1N1)pdm09 influenza virus (8), there is no clinical data indicating the involvement of a specific protease in the activation of seasonal IAV. However, in mice, TMPRSS2 has been identified as being essential for the spread of H1N1 IAV (9)(10)(11) and of H3N2 IAV in association with TMPRSS4 (12).…”

Transcriptome analysis reveals TMPRSS6 isoforms with distinct functionalities

“…Human type II transmembrane serine proteases (TTSPs) are a family of enzymes that consists of 17 members sharing common structural features and are considered key players of cell surface proteolysis . Many TTSPs have been associated with biological processes and pathologies, which positions them as attractive therapeutic targets for diseases, such as viral infection and cancers …”

In vitro methods for testing antiviral drugs

“…This triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. Some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (Laporte and Naesens, 2017)] such as influenza virus (Böttcher-Friebertshäuser et al, 2010;Kühn et al, 2016), Newcastle disease virus (Gotoh et al, 1992), and respiratory syncytial virus (Sugrue et al, 2001). Extensive research efforts have yielded detailed information about HIV-1 protease and its inhibitors [reviewed in (De Clercq, 2004;Konvalinka et al, 2015;Midde et al, 2016)].…”