The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses
- Kouji Sakaia,
- Yasushi Amib,
- Maino Taharaa,
- Toru Kubotaa,
- Masaki Anrakua,
- Masako Abea,
- Noriko Nakajimac,
- Tsuyoshi Sekizukad,
- Kazuya Shiratoa,
- Yuriko Suzakib,
- Akira Ainaie,
- Yuichiro Nakatsua,
- Kazuhiko Kanouf,
- Kazuya Nakamurae,
- Tadaki Suzukic,
- Katsuhiro Komasea,
- Eri Nobusawae,
- Katsumi Maenakag,
- Makoto Kurodad,
- Hideki Hasegawac,
- Yoshihiro Kawaokah,i,j,
- Masato Tashiroe and
- Makoto Takedaa
- aDepartment of Virology 3, National Institute of Infectious Diseases, Tokyo, Japan
- bDivision of Experimental Animal Research, National Institute of Infectious Diseases, Tokyo, Japan
- cDepartment of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
- dLaboratory of Bacterial Genomics, Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan
- eInfluenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan
- fInfectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan
- gLaboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
- hDivision of Virology, Department of Microbiology and Immunology, and International Research Center for Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- iERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan
- jInfluenza Research Institute, University of Wisconsin—Madison, Madison, Wisconsin, USA
- T. S. Dermody, Editor
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FIG 1Proteolytic activation of HA by TMPRSS2. (A) HA proteins (H1, H3, H5, and H7 subtypes) were expressed alone (−) or together with mTMPRSS2 (m) or hTMPRSS2 (h) in 293T cells using expression plasmids. The cells were pulse labeled, and the HA components (HA0, HA1, and HA2) were detected and analyzed by immunoprecipitation and SDS-PAGE. (B) HA proteins were expressed in HeLa/mTMPRSS2, HeLa/hTMPRSS2, or parental HeLa (−) cells. At 2 days posttransfection, the cells were treated with low-pH buffer (pH 5.3), and cell-cell fusion was analyzed by immunofluorescence staining using anti-IAV antibodies coupled with Alexa Fluor 488- or 549-conjugated secondary antibodies. The nuclear DNA was stained with DAPI.
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FIG 2Structure of the targeted TMPRSS2 gene. (A) TMPRSS2 KO mice possess an allele [Tmprss2tm1(KOMP)Vlcg] with an ablating deletion of the TMPRSS2 gene, which was replaced by the lacZ gene (VelociGene KOMP definitive null allele design). (B) The genotype was analyzed by VelociGene KOMP allele PCR genotyping strategies using primers NeoFwd and SD and a previously reported method using primers P11 and P12 (25).
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FIG 3Role of TMPRSS2 in H1N1 and H3N2 IAV pathogenicity. (A and B) WT and TMPRSS2 KO mice were intranasally inoculated with different doses of MA-CA04[H1N1] (A) or MA-GZX[H3N2] (B) (n = 4 to 7). Body weights were measured daily. Error bars represent standard deviations. (C and D) Survival curves of IAV-infected mice. WT and TMPRSS2 KO mice were challenged with different doses of MA-CA04[H1N1] (C) or MA-GZX[H3N2] (D). For each experimental group, 4 to 6 mice were used. (E to H) Histopathological findings in the lungs of WT and TMPRSS2 KO mice infected with MA-CA04[H1N1] (E and G) or MA-GZX[H3N2] (F and H). Data obtained by hematoxylin and eosin staining (magnification, ×10) (E and F) and immunohistochemistry for the IAV nucleocapsid protein (magnification, ×10) (G and H) are shown. The inflammation scores of individual mice (n = 3) are shown at the bottom of each panel (E and F): 0, no apparent changes; 1, minimal changes or bronchiolitis; 2, bronchiolitis and/or slight alveolitis; 3, mild alveolitis with neutrophils, monocytes/macrophages, or lymphocytes; 4, moderate alveolitis.
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FIG 4Proteolytic activation of IAVs in vivo. (A and B) WT and TMPRSS2 KO mice were intranasally inoculated with PBS (mock) (n = 1) or MA-GZX[H3N2] (n = 3). Lung lavage fluids (A) and lung homogenates (B) were collected at 6 dpi, and HA was analyzed by SDS-PAGE and immunoblotting. Each lane corresponds to data from an individual mouse. (C and D) WT and TMPRSS2 KO mice were intranasally inoculated with MA-CA04[H1N1] (n = 3) or MA-GZX[H3N2] (n = 3). Lung lavage fluids at 2, 4, and 6 dpi (C) and lung homogenates at 2 dpi (D) were either untreated (Trypsin −) or treated with trypsin (Trypsin +) and used for virus titration. Error bars represent standard deviations.
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FIG 5Role of TMPRSS2 in H5N1 and H7N9 IAV pathogenicity. (A and B) WT and TMPRSS2 KO mice were intranasally inoculated with different doses of Anhui1[H7N9] (A) or VN1194[H5N1] (B) (n = 4 to 6). The body weights were measured daily. Error bars represent standard deviations. (C and D) Survival curves of IAV-infected mice. WT and TMPRSS2 KO mice were challenged with different doses of Anhui1[H7N9] (C) or VN1194[H5N1] (D). For each experimental group, 4 to 7 mice were used. (E and F) WT and TMPRSS2 KO mice were intranasally inoculated with Anhui1[H7N9] (n = 3) or VN1194[H5N1] (n = 3). Lung homogenates at 4 dpi (D) were either untreated (Trypsin −) or treated with trypsin (Trypsin +) and used for virus titration. Error bars represent standard deviations. (G to J) Histopathological findings in the lungs of WT and TMPRSS2 KO mice infected with Anhui1[H7N9] (G and I) or VN1194[H5N1] (H and J). Data obtained by hematoxylin and eosin staining (magnification, ×10) (G and H) and immunohistochemistry for the IAV nucleocapsid protein (magnification, ×10) (I and J) are shown. The inflammation scores of individual mice (n = 3) are shown at the bottom of each panel (G and H): 0, no apparent changes; 1, minimal changes or bronchiolitis; 2, bronchiolitis and/or slight alveolitis; 3, mild alveolitis with neutrophils, monocytes/macrophages, or lymphocytes; 4, moderate alveolitis.
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