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. 2021 Oct 13;95(21):e0129621.
doi: 10.1128/JVI.01296-21. Epub 2021 Aug 18.

TMPRSS2 Activates Hemagglutinin-Esterase Glycoprotein of Influenza C Virus

Ko Sato  1   2   3 , Hideki Hayashi  4 , Yoshitaka Shimotai  5 , Mutsuo Yamaya  6 , Seiji Hongo  5 , Kazuyoshi Kawakami  2   3 , Yoko Matsuzaki  5 , Hidekazu Nishimura  1
Affiliations

TMPRSS2 Activates Hemagglutinin-Esterase Glycoprotein of Influenza C Virus

Ko Sato et al. J Virol. .

Abstract

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV and IBV, respectively). It has a monobasic site, which is cleaved by some host enzymes. The cleavage is essential to activating the virus, but the enzyme or enzymes in the respiratory tract have not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK cells (MDCK-TMPRSS2 and MDCK-HAT). ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multistep growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs for ICV infection. IMPORTANCE Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, which possesses receptor-binding, receptor-destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme or enzymes in the respiratory tract have not been identified. This study revealed that transmembrane protease serine S1 member 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of ICV infection.

Keywords: HAT; HE; TMPRSS2; influenza C virus; serine protease.

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Figures

FIG 1
FIG 1
mRNA and protein expression of each serine protease in MDCK-TMPRSS2 and MDCK-HAT cells. (A) Real-time RT-PCR-quantified expression levels of tmprss2 and tmprss11d mRNA in MDCK, MDCK-TMPRSS2, and MDCK-HAT cells. Each column represents the mean ± SD from triplicate cultures. ND, not detected. (B) Levels of protein expression of TMPRSS2 and HAT (also known as TMPRSS11D) in MDCK, MDCK-TMPRSS2, and MDCK-HAT cells were analyzed by indirect immunofluorescence using specific antibodies and Alexa Fluor 546 (orange)-conjugated secondary antibodies. Nuclei were stained with Hoechst 33342 (blue). Size bars, 20 μm.
FIG 2
FIG 2
Analysis of viral replication and spike protein cleavage in MDCK-TMPRSS2 and MDCK-HAT cells. (A) Each MDCK cell was infected with ICV (C/Ann Arbor/1/50) and IAV (A/Sendai-H/N633/09) at an MOI of 0.01 and then cultured with or without 5 μg/ml trypsin. Viral titers in these supernatants were measured by TCID50 endpoint assay. Each symbol represents the mean ± SD from three independent experiments. HE and HA glycoproteins in the supernatants (B) and whole-cell lysates (C) of each MDCK cells at 48 and 24 h postinfection with ICV (C/Ann Arbor/1/50) and IAV (A/Sendai-H/N633/09) at MOI of 10, respectively, in the presence or absence of 5 μg/ml trypsin were detected by Western blotting. The cleavage rates of HE and HA were quantified from the band density using Image Lab software: % HE cleavage = [HE1/(HE1 + HE0)] × 100, and % HA cleavage = [HA1/(HA1 + HA0)] × 100. Each column represents the mean ± SD from three independent experiments. Significant difference in MDCK cell culture without trypsin: *, P < 0.05; **, P < 0.01; ***, P < 0.005. NS, not significant. hpi, hour postinoculation.
FIG 3
FIG 3
Analysis of viral replication in MDCK-TMPRSS2 and MDCK-HAT cells. Each MDCK cell was infected with ICV (C/Yamagata/8/2002) at an MOI of 0.01 and then cultured with or without 5 μg/ml trypsin. Viral titers in these supernatants were measured by TCID50 endpoint assay. Each symbol represents the mean ± SD from three independent experiments. Significant difference in MDCK cell culture without trypsin: **, P < 0.01; ***, P < 0.005.
FIG 4
FIG 4
Analysis of serine protease inhibitors for viral replication in MDCK cells. MDCK-TMPRSS2 and MDCK-HAT cells were infected with ICV (C/Ann Arbor/1/50) and IAV (A/Sendai-H/N633/09) at an MOI of 0.01 in the presence of camostat or nafamostat at 10 μg/ml (20 and 19 μM, respectively). Viral titers in these supernatants at 7 days postinoculation were measured by TCID50 endpoint assay. Each column represents the mean ± SD from three independent experiments. NS, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.005.
FIG 5
FIG 5
Analysis of serine protease inhibitors for viral replication in HNE cells. (A) Expression levels of tmprss2 and tmprss11d mRNA in HNE cells were quantified by real-time RT-PCR. Each column represents the mean ± SD from triplicate cultures. (B) HNE cells were infected with ICV (C/Ann Arbor/1/50) and IAV (A/Sendai-H/N633/09) at an MOI of 0.1 in the presence of camostat or nafamostat at the indicated dose, and viral titers in these supernatants at 7 days postinoculation were measured. Each symbol represents the mean ± SD from three independent experiments. Significant difference for inhibitors at 0 μg/ml: *, P < 0.05; **, P < 0.01; ***, P < 0.005. Significant difference between the inhibitors: ‡, P < 0.05; ‡‡, P < 0.01.
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