The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses
ABSTRACT
Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2+/+ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with ≥1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo.
IMPORTANCE Influenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs in vivo through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2 in vitro. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health.
INTRODUCTION
Influenza A virus (IAV) is classified in the Orthomyxoviridae family and is a leading agent that affects and kills humans worldwide. IAV enters target cells via endocytosis, and virus-cell membrane fusion occurs at the late endosomes, thus releasing the viral genome to start virus replication. Membrane fusion is mediated by the hemagglutinin (HA) protein, which is synthesized as the inactive precursor HA0 and cleaved by a host cell protease(s) to gain fusion activity. Proteolytic cleavage of HA0 into the HA1 and HA2 subunits is essential for HA to express membrane fusion activity and, consequently, for IAV to acquire infectivity.
The HA of low-pathogenic (LP) IAVs, for which infection remains localized at respiratory and/or alimentary epithelial cells in birds and mammals, including humans, possesses a single arginine residue at the cleavage site. The HA of LP IAVs is thought to be cleaved and activated by a specific protease(s) present exclusively in these tissues. Trypsin, miniplasmin, tryptase Clara, mast cell tryptase, and type II transmembrane serine proteases (TTSPs) such as TMPRSS2, human airway trypsin-like protease (HAT), TMPRSS4, and matriptase have been shown to cleave the HA of LP IAVs at the single arginine residue (1, 2). Previous studies have also demonstrated HA subtype and strain specificities of TTSPs (3–6). On the other hand, the HA of high-pathogenic (HP) IAVs such as HP H5N1, which causes fatal systemic infections, contains multiple basic amino acids at the cleavage site (7–9). The HA of HP IAVs is cleaved by ubiquitous intracellular proteases such as furin and proprotein convertase 5/6, which are present in all cell types (1, 2). The concept of host protease-dependent tissue tropism and pathogenicity of LP and HP IAVs has been well established (7–9). However, the protease(s) directly involved in the cleavage activation of each HA subtype remains to be identified.
In the present study, we focused primarily on TMPRSS2 because this protease is expressed in the respiratory tract, activates a variety of respiratory viruses, and cleaves the HA of IAVs efficiently, even at marginal levels of expression, in vitro (4, 10–16). Our working hypothesis was that if TMPRSS2 is essential for IAV activation in vivo, mice lacking TMPRSS2 expression (TMPRSS2 knockout [KO] mice) are highly tolerant of challenge infection by IAVs that use primarily TMPRSS2 for HA cleavage.
MATERIALS AND METHODS
Ethics statement.
All experiments with animals were performed in strict accordance with the animal experimentation guidelines of the National Institute of Infectious Diseases. The protocol was approved by the Institutional Animal Care and Use Committee of the institute (permit numbers 113066-II, 313008-II, and 13-09).
Plasmids.
A mouse TMPRSS2 (mTMPRSS2) expression plasmid, pTarget-mTMPRSS2, was generated by inserting the mTMPRSS2 cDNA obtained from a C57BL/6 mouse. The human TMPRSS2 (hTMPRSS2) expression plasmid pcDNA-TMPRSS2 was reported previously (11). IAV HA expression plasmids pCAGGS-H1, -H3, -H5, and -H7 were generated by inserting cDNAs of the HA-coding region from mouse-adapted (MA) A/California/04/09 (MA-CA04[H1N1]) (17), MA-A/Guizhou-X (MA-GZX[H3N2]) (18), A/Vietnam/1194/04 (VN1194[H5N1]) (19), and A/Anhui/1/2013 (Anhui1[H7N9]) (20), respectively.
Cells and viruses.
MDCK, HeLa, and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). HeLa cells were transfected with pTarget-mTMPRSS2 or pcDNA-TMPRSS2, and Geneticin-resistant stable clones (HeLa/mTMPRSS2 and HeLa/hTMPRSS2, respectively) were selected in the presence of 1.0 mg/ml of Geneticin (G418; Nacalai Tesque). MA-CA04[H1N1] was generated by reverse genetics as reported previously (17). MA-GZX[H3N2], VN1194[H5N1], and Anhui1[H7N9] were reported previously (18–20).
Structural modeling of hTMPRSS2 and mTMPRSS2.
No known structures of mTMPRSS2 or hTMPRSS2 are available. Therefore, three-dimensional structural models of hTMPRSS2 and mTMPRSS2 were generated by a homology modeling method using several protease structures as the templates. The I-TASSER modeling server (21) was used for modeling. The X-ray structure most similar to both TMPRSS2 models was the human transmembrane serine protease hepsin (PDB accession number 1Z8G). The models were refined by energy minimization using Swiss-PdbViewer (22) and finally evaluated by PROCHECK (23) and Verify3D (24).
Cell fusion assay.
HeLa/mTMPRSS2, HeLa/hTMPRSS2, and parental HeLa cells were transfected with an HA-expressing plasmid (pCAGGS-H1, -H3, -H5, or -H7) by using X-tremeGENE HP DNA transfection reagent (Roche). At 2 days posttransfection, the cells were washed with phosphate-buffered saline (PBS) and treated with prewarmed low-pH buffer (145 mM NaCl, 20 mM sodium citrate [pH 5.3]) for 2 min. The low-pH buffer was then replaced with DMEM–5% FCS, and the cells were incubated at 37°C for 3 h. Cell-cell fusion was analyzed by immunofluorescence staining using anti-IAV rabbit serum against H7, anti-IAV goat serum against H3, and HA-specific rabbit monoclonal antibodies (clone 327 for H1 and clone 89 for H5) coupled with Alexa Fluor 488- or 549-conjugated secondary antibodies. The nuclear DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI).
Pulse labeling and immunoprecipitation.
Monolayers of 293T cells were transfected with an HA-expressing plasmid (pCAGGS-H1, -H3, -H5, or -H7) alone or together with a TMPRSS2-expressing plasmid (pTarget-mTMPRSS2 or pcDNA-TMPRSS2). At 20 h posttransfection, the cells were cultured in methionine-cysteine-deficient medium for 1 h and then pulse labeled with [35S]methionine-cysteine by using EasyTag EXPRE35S35S protein labeling mix (PerkinElmer) for 1 h. The cells in some wells were treated with 1 μg/ml acetylated trypsin (Sigma) for 1 h, as a trypsin-activated HA control, and all cells were lysed in radioimmunoprecipitation assay (RIPA) buffer. Polypeptides in the cell lysates were immunoprecipitated with anti-IAV rabbit sera against H3 and H7 (Sino Biological Inc.) and HA-specific rabbit monoclonal antibodies (clone 327 for H1 and clone 89 for H5) and analyzed by SDS-PAGE.
Generation of TMPRSS2 KO mice.
TMPRSS2 gene KO C57BL/6 embryonic stem (ES) cells (product number VG13341) were obtained from the Knockout Mouse Project (KOMP) Repository (UC Davis). The ES cells possessed 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). The ES cells were injected into C57BL/6 mouse blastocysts, and chimeric mice in a complete C57BL/6 genetic background were generated. Male chimeric mice were selected by VelociGene KOMP allele PCR genotyping strategies by using primers NeoFwd and SD and a previously reported method using primers P11 and P12 (25) and mated with normal female C57BL/6 mice to generate C57BL/6 mice with a heterozygous genotype of the TMPRSS2 gene (TMPRSS2+/−). TMPRSS2 KO mice with a homologous genotype (TMPRSS2−/−) were obtained by crossing male and female TMPRSS2+/− C57BL/6 mice.
Transcriptome analysis.
The bronchi and right middle lobes of the lung were obtained from uninfected healthy wild-type (WT) and KO (TMPRSS2−/−) mice (6-week-old females). Total RNA was prepared from ∼50 mg of the bronchi or right middle lobes treated with RNAlater (Life Technologies), using a RecoverAll Total Nucleic Acid Isolation kit (Ambion), followed by selection of poly(A) mRNA using a FastTrack MAG mRNA isolation kit (Invitrogen). RNA strand-specific transcriptome sequencing (RNA-seq) libraries were prepared from ∼50 ng of the purified poly(A) mRNA using a ScriptSeq v2 RNA-Seq Library Preparation kit (Epicentre). The prepared RNA-seq library was sequenced by using a GAIIx sequencer (Illumina). The obtained strand-specific short reads were analyzed by CLC Genome Workbench (v. 6.05) with mean expression normalization against Mus musculus reference genome sequences (GRCm38.p1), and genes showing significant differential expression were detected with a false discovery rate (FDR) of <0.05 and changes of ≥2-fold. The reads per kilobase of exon model per million mapped reads (RPKM) were estimated for each transcriptome experiment.
Titration of infectious units.
To determine the infectious virus titers activated in vivo, monolayers of MDCK cells in 12-well plates were infected with serially diluted virus samples for 1 h at 4°C, washed twice with PBS, overlaid with DMEM–1% agarose, and incubated for 24 h at 37°C to allow the viruses to enter the cells. Trypsin was omitted to avoid HA cleavage before virus entry. At 24 h postinfection, the cell monolayers were additionally overlaid with DMEM–1% agarose supplemented with 2.0 μg/ml of trypsin to allow plaque formation. The virus titers obtained with this method were essentially equivalent to those obtained by immunofluorescence staining techniques (26), in which trypsin was omitted during the entire process of titration and individual infected cells, instead of plaques, were detected and counted. To determine the infectious titers of viruses that had not been activated in vivo but possessed infectious potential, virus samples were treated with 1.0 μg/ml of trypsin and subjected to a standard plaque assay.
Infection of mice.
KO (TMPRSS2−/−) and WT (TMPRSS2+/+) C57BL/6 mice (6- to 7-week-old males or females) were challenged with MA-CA04[H1N1], MA-GZX[H3N2], VN1194[H5N1], or Anhui1[H7N9]. For each mouse, 20 μl of virus solution containing different amounts of IAV was inoculated intranasally. For mock infection, the same volume of PBS was used. For each experimental group, 4 to 6 mice were used to monitor body weight. To analyze the in vivo activation and replication of IAV, WT and TMPRSS2 KO mice were challenged with 4.0 × 103 PFU of MA-CA04[H1N1] or 6.8 × 103 PFU of MA-GZX[H3N2]. Lung lavage fluids collected at 2, 4, and 6 days postinfection (dpi) and lung homogenates collected at 2 dpi were either untreated or treated with trypsin to determine the virus titers activated in vivo and in vitro, respectively. Three mice were used for each experimental group, and samples were collected separately from individual mice. For histopathological examinations, WT and TMPRSS2 KO mice were infected with 4.0 × 103 PFU (3.0 × 102 50% lethal doses [LD50]) of MA-CA04[H1N1], 6.8 × 103 PFU (1.0 × 102 LD50) of MA-GZX[H3N2], 1.0 × 104 PFU (1.0 × 102 LD50) of VN1194[H5N1], or 4.0 × 106 PFU (1.0 × 102 LD50) of Anhui1[H7N9]; euthanized; and autopsied at 2 and 6 dpi. Three mice were used for each experimental group.
Immunoblotting.
The lung lavage fluids and lung homogenates were mixed with lysis buffer to make a final solution containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 4 mM EDTA, 0.1% sodium deoxycholate, 1% Nonidet P-40, and 0.1% SDS, containing a complete protease inhibitor mixture (Roche Diagnostics). The polypeptides were then separated by SDS-PAGE and immunoblotted. A rabbit antiserum raised against H3 (Sino Biological Inc.) was used for detection.
Nucleotide sequence accession number.
The RNA-seq reads of the lung tissues from WT and TMPRSS2 KO mice are available at the DDBJ Sequence Read Archive under accession number DRA001103 (47,935,571 and 46,863,938 paired-end reads for WT and TMPRSS2 KO mice, respectively).
RESULTS
mTMPRSS2 proteolytically activates IAV HAs similarly to hTMPRSS2.
To obtain a biological rationale for performing mouse experiments, we analyzed mTMPRSS2. Three-dimensional structural models of mTMPRSS2 and hTMPRSS2 showed strong conservation of amino acid residues near the protease active site (data not shown). Based on the strong homology between hTMPRSS2 and mTMPRSS2, these proteases were strongly suggested to have similar substrate specificities. However, the TMPRSS2 structures were strictly models, and biological analyses were performed. The analyses showed that all HAs of LP IAVs of the H1, H3, and H7 subtypes were cleaved by both mTMPRSS2 and hTMPRSS2 (Fig. 1A), and the cleaved HAs showed cell-cell fusion (syncytium formation) activity (Fig. 1B). As expected, cleavage and activation of HA of an HP IAV of the H5 subtype occurred independently of TMPRSS2 expression (Fig. 1A and B). Consistent with previous data (27), different molecular sizes of HA were detected in TMPRSS2-expressing cells, because the expression of TMPRSS2 modulates the HA glycosylation pattern (Fig. 1A).
FIG 1
TMPRSS2 is essential for the pathogenicity of H1N1 and H3N2 IAVs.
TMPRSS2 KO mice, which possessed an ablating deletion including exons 3 to 13 of the TMPRSS2 gene, were generated in a complete C57BL/6 genetic background (Fig. 2). To confirm whether TMPRSS2 KO induced unexpected differential expression of other TTSPs, RNA-seq experiments were performed. The transcriptome analysis indicated that TMPRSS2 KO did not significantly affect the expressions of other TTSP genes and potential protease genes (Table 1). As an exception, the level of St14 appeared to be increased in the TMPRSS2 KO mouse lung (Table 1), but the raw count was still very low, and the change was judged to be insignificant based on the FDR analysis. However, our data did not exclude the possibility that the change was induced by TMPRSS2 gene KO. TMPRSS2 KO mice exhibited normal reproduction, development, and growth patterns, similarly to WT C57BL/6 mice, as observed for previously reported TMPRSS2 KO mice with a deletion of exons 10 to 13 in the TMPRSS2 gene (25).
FIG 2
TABLE 1
| Gene | RPKM | |||
|---|---|---|---|---|
| Lungs | Bronchi | |||
| WT | KO | WT | KO | |
| TTSPs | ||||
| Tmprss2 | 2.43 | 0.59a | 3.62 | 0.51a |
| Tmprss3 | 0.00 | 0.00 | 0.21 | 0.00 |
| Tmprss4 | 0.54 | 0.64 | 0.61 | 0.40 |
| Tmprss5 | 0.05 | 0.21 | 1.61 | 0.38 |
| Tmprss6 | 0.01 | 0.00 | 0.02 | 0.05 |
| Tmprss7 | 0.39 | 0.00 | 0.01 | 0.03 |
| Tmprss9 | 0.00 | 0.00 | 0.03 | 0.02 |
| Tmprss11a | 0.17 | 0.00 | 0.42 | 0.13 |
| Tmprss11bnl | 0.32 | 0.00 | 0.00 | 0.00 |
| Tmprss11c | 0.09 | 0.32 | 0.00 | 0.00 |
| Tmprss11d | 0.53 | 0.00 | 0.00 | 0.00 |
| Tmprss11e | 0.07 | 0.00 | 0.00 | 0.00 |
| Tmprss11f | 0.03 | 0.00 | 0.00 | 0.00 |
| Tmprss11g | 0.01 | 0.02 | 0.00 | 0.00 |
| Tmprss12 | 0.00 | 0.00 | 0.00 | 0.00 |
| Tmprss13 | 0.03 | 0.00 | 2.22 | 1.46 |
| Tmprss15 | 0.01 | 0.02 | 0.11 | 0.07 |
| Hpn | 0.00 | 0.00 | 2.23 | 3.11 |
| Lrp4 | 9.43 | 15.75 | 1.77 | 2.23 |
| St14 | 0.35 | 4.35 | 12.96 | 13.74 |
| Internal controls | ||||
| Gapdh | 31.06 | 86.26 | 441.28 | 794.44 |
| Actb | 264.31 | 491.48 | 308.25 | 296.38 |
a
Detected reads were mapped to exon 14 in the Tmprss2 coding sequence remaining in the TMPRSS2 KO mouse genome.
Various doses of MA viruses of human LP IAV strains MA-CA04[H1N1] and MA-GZX[H3N2] were inoculated intranasally into WT and TMPRSS2 KO mice. All WT mice inoculated with ≥102 PFU (7.5 LD50) of MA-CA04[H1N1] died or required euthanasia (Fig. 3A and C). In strong contrast, TMPRSS2 KO mice challenged with up to 105 PFU (7.5 × 103 LD50) showed neither clinical signs nor body weight loss (Fig. 3A and C). TMPRSS2 KO mice were also highly tolerant of lethal challenge infections with MA-GZX[H3N2] (Fig. 3B and D). All TMPRSS2 KO mice infected with 105 PFU (1.5 × 103 LD50) of MA-GZX[H3N2] showed moderate body weight loss but recovered completely (Fig. 3B and D).
FIG 3
Figure 3E to H shows the histopathology of lungs of mice infected with 4.0 × 103 PFU (3.0 × 102 LD50) of MA-CA04[H1N1] or 6.8 × 103 PFU (1.0 × 102 LD50) of MA-GZX[H3N2]. WT mice infected with each virus developed bronchiolitis and peribronchial inflammation, with a few foci of alveolitis at 2 dpi and mild to moderate alveolitis at 6 dpi (Fig. 3E and F). Viral antigens were detected in bronchial epithelial cells and alveolar lining cells in the lungs of WT mice at 2 dpi and had spread dramatically to the entire lungs by 6 dpi (Fig. 3G and H). In contrast, the lungs of KO mice infected with the same viruses exhibited significantly reduced levels of inflammation (Fig. 3E and F). Viral antigen-positive cells did not spread during the observation period (Fig. 3G and H). Viral antigens were almost completely eliminated from bronchiolar epithelial cells of KO mice by 6 dpi (Fig. 3G and H).
TMPRSS2 is essential for proteolytic activation of IAVs in vivo.
To explore the cleavage of HA in the lungs, WT and TMPRSS2 KO mice were infected with 4.0 × 103 PFU (3.0 × 102 LD50) of MA-CA04[H1N1] or 6.8 × 103 PFU (1.0 × 102 LD50) of MA-GZX[H3N2]. At 6 dpi, lung lavage fluids were collected from mice infected with MA-GZX[H3N2] and analyzed for HA protein by SDS-PAGE and immunoblotting. Cleavage of HA was demonstrated in WT mice, as signals for the HA1 and HA2 subunits were clearly detected in these mice (Fig. 4A). In contrast, only the HA0 precursor was detected in TMPRSS2 KO mice, and HA1 and HA2 remained undetectable (Fig. 4A). Similarly, lung homogenates of the infected mice were analyzed. HA was efficiently cleaved into HA1 and HA2 in the lung homogenates of WT mice (Fig. 4B). Meanwhile, the lung homogenates of TMPRSS2 KO mice showed HA0 signals, but HA1 and HA2 were undetectable (Fig. 4B). These findings indicated that the HA protein of progeny viruses present in the respiratory tract had been cleaved in WT mice, while HA cleavage was severely impaired in TMPRSS2 KO mice. To verify whether the infectivity of the progeny viruses from each mouse lung was activated in vivo, lung lavage fluids at 2, 4, and 6 dpi and lung homogenates at 2 dpi were treated or untreated with trypsin in vitro and compared for infectivity in vitro to determine virus titers. At 2 dpi, the virus titers of MA-CA04[H1N1] and MA-GZX[H3N2] reached their peak levels in WT mice (Fig. 4C). The HAs of progeny viruses were shown to be fully activated in WT mice, since the virus infectivity titers without trypsin treatment (Fig. 4C and D) were similar to those after activation in vitro (Fig. 4C and D). The virus titers in the lungs of TMPRSS2 KO mice were much lower than those in the lungs of WT mice (Fig. 4C and D), showing restricted virus growth in TMPRSS2 KO mice. Importantly, the virus titers (Fig. 4C and D) were further increased 10 to 100 times after trypsin treatment in vitro (Fig. 4C and D). These data demonstrated that the great majority (90 to 99%) of the progeny virus particles failed to be proteolytically activated in TMPRSS2 KO mice.
FIG 4
TMPRSS2 is essential for LP H7N9 IAV, but dispensable for HP H5N1 IAV, to exhibit pathogenicity.
The pathogenicity of a human isolate of an emerging LP H7N9 subtype virus, Anhui1[H7N9], was also analyzed. Anhui1[H7N9] was inoculated intranasally into WT and TMPRSS2 KO mice. As shown in Fig. 5A and C, TMPRSS2 KO mice were highly tolerant of H7N9 virus infection. All WT mice infected with 105 PFU (5.0 × 10 LD50) of Anhui1[H7N9] died or required euthanasia by 8 dpi (Fig. 5A and C). In contrast, all TMPRSS2 KO mice infected with the same dose (105 PFU [5.0 × 10 LD50]) showed no clinical signs, and those infected with a 1,000-times-higher dose, 108 PFU (5.0 × 104 LD50), showed only temporary and mild body weight loss (Fig. 5A). The virus titers in the KO mouse lungs were much lower than those in the WT mouse lungs (Fig. 5E). Histopathological examinations of mouse lungs infected with 4.0 × 106 PFU of Anhui1[H7N9] showed that Anhui1[H7N9] spread poorly in the lungs of TMPRSS2 KO mice (Fig. 5G and I).
FIG 5
In contrast to the findings for the LP IAVs described above, a human isolate of an HP H5N1 subtype virus, VN1194[H5N1], caused severe body weight loss, neurological symptoms, and death or requirement for euthanasia by 8 or 9 dpi in both WT and TMPRSS2 KO mice infected with 104 PFU (2.0 × 103 LD50) of VN1194[H5N1] (Fig. 5B and D). The survival curves of WT and TMPRSS2 KO mice infected with 102 PFU (5.0 × 10 LD50) of VN1194[H5N1] were also similar to one another (Fig. 5B and D). The virus titers in the lungs were equivalent between WT and KO mice (Fig. 5F). Histopathological examinations of mouse lungs infected with 1.0 × 104 PFU of VN1194[H5N1] demonstrated that VN1194[H5N1] spread efficiently in a similar manner in the lungs of WT and TMPRSS2 KO mice (Fig. 5H and J). The overall results indicated that, unlike LP IAVs, proteolytic activation of the HP H5N1 virus occurred in mouse lungs independently of TMPRSS2.
DISCUSSION
In the last decade, we have experienced outbreaks of a swine-origin LP H1N1 IAV and the severe acute respiratory syndrome (SARS) coronavirus (28, 29). These outbreaks proved that respiratory viruses can currently spread rapidly and that the available strategies are insufficiently effective to prevent global transmission of emerging respiratory viruses (28). At this time, H5N1 and H7N9 IAVs and the Middle East respiratory syndrome (MERS) coronavirus are potential threats for humans (29, 30).
The present study has provided concrete evidence that TMPRSS2 expression is essential for in vivo replication of emerging H7N9 and seasonal IAVs and that this protease primarily determines IAV pathogenicity. Based on the strong homology between hTMPRSS2 and mTMPRSS2, hTMPRSS2 is strongly suggested to play a similar role in the activation and pathogenicity of human IAVs in vivo. In human airway bronchial epithelial cells, TMPRSS2 is expressed by type 1 and type 2 pneumocytes of the alveolar epithelium and alveolar macrophages (12, 31). In the airway, IAV particles are released exclusively from the apical membrane (32). To achieve this, the IAV HA protein is specifically targeted to the apical plasma membrane (33, 34), and TMPRSS2 is selectively expressed at the apical membrane of airway epithelial cells (35). Taking these data into consideration, it is most likely that LP IAVs use primarily the specific host protease TMPRSS2 for HA activation in the lungs. However, our data do not exclude the possibility that TMPRSS2 may activate a precursor of another protease(s) that finally induces HA activation. Nevertheless, our data showing the critical role of TMPRSS2 expressed in the respiratory tract in the activation of IAV pathogenicity in vivo support the concept of host protease-mediated pathogenicity of IAVs (7–9).
The present findings suggest that TMPRSS2 is a good target for the development of anti-LP IAV drugs. The membrane-anchoring and cytoplasmic domains of TTSPs regulate cellular trafficking and localization of TTSPs, thereby enabling temporal and spatial regulation of substrate processing (36–38). Accordingly, by targeting specific substrates such as peptide hormones, growth factors, differentiation factors, receptors, and adhesion molecules, TTSPs play a variety of critical roles in developmental stages and physiological events (36–38). A previous study suggested that TMPRSS2 regulates Na+ currents (39). However, the physiological roles of TMPRSS2 remain to be elucidated (40), because TMPRSS2 KO mice showed a normal phenotype.
During the submission process for the present paper, Hatesuer et al. (41) reported a similar study demonstrating a critical role of TMPRSS2 in the pathogenicity of H1N1 and H3N2 IAVs in mice. They used a previously reported TMPRSS2 KO mouse strain with a deletion of exons 10 to 13 in the TMPRSS2 gene (25, 41). The TMPRSS2 KO mice with a deletion of exons 3 to 13 in the TMPRSS2 gene established in the present study exhibited phenotypes very similar to those of their mice, providing a very strong conclusion that TMPRSS2 expression is essential for IAV pathogenesis. Interestingly, in agreement with their data (41), our study demonstrated that replication of H1N1 IAV was more severely restricted in TMPRSS2 KO mice than that of H3N2 IAV. Therefore, cleavage of the H1 subtype appears to be more strongly dependent on TMPRSS2 expression than cleavage of the H3 subtype.
Finally, TTSPs have a clear substrate specificity (37). Nevertheless, various respiratory viruses, including the SARS coronavirus (12, 42–44), MERS coronavirus (14), human metapneumovirus (11), and parainfluenza viruses (13), also use TMPRSS2 for their activation. These observations imply that a variety of respiratory viruses may specifically exploit TMPRSS2 for activation. Drugs that inhibit TMPRSS2 are therefore expected to be effective against a wide spectrum of respiratory viruses. Meanwhile, different types of drugs targeting ubiquitous proteases other than TMPRSS2 may be required for HP IAVs such as the H5N1 virus subtype.
ACKNOWLEDGMENTS
The ES cells used for this research project were generated by the Trans-NIH Knockout Mouse Project (KOMP) and obtained from the KOMP Repository (http://www.komp.org/). NIH grants to VelociGene at Regeneron Inc. (U01HG004085) and the CSD Consortium (U01HG004080) funded the generation of gene-targeted ES cells for 8,500 genes in the KOMP Program, which were archived and distributed by the KOMP Repository at UC Davis and CHORI (U42RR024244). This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (KAKENHI; 23390114), the Takeda Science Foundation, and ERATO, Japan, and by a National Institute of Allergy and Infectious Diseases Public Health Service research grant.
We thank Yuelong Shu, Chinese Center for Disease Control and Prevention, and Le Quynh Mai, National Institute of Hygiene and Epidemiology, for providing H7N9 IAV and H5N1 IAV, respectively; R. A. Lamb, Northwestern University, for providing anti-IAV serum; and E. Takashita, M. Shirakura, H. Asanuma, H. Fukuhara, A. Sato, N. Nagata, Y. Sato, and all our members in the Department of Virology 3, NIID, for suggestions and technical support.
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Information & Contributors
Information
Published In
Journal of Virology
Volume 88 • Number 10 • 15 May 2014
Pages: 5608 - 5616
Editor: T. S. Dermody
PubMed: 24600012
Copyright
© 2014 American Society for Microbiology.
History
Received: 11 December 2013
Accepted: 27 February 2014
Published online: 15 May 2014
Contributors
Editor
T. S. Dermody
Editor
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Citation
2014.
The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses. J Virol
88:.
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