Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts

Abstract

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.

Figure 1. Proteolytic activation of influenza virus hemagglutinin and SARS spike protein is conserved between TMPRSS2 of human, porcine, avian and murine origin.

(A) Expression plasmids encoding the HA of the 1918 influenza virus and the indicated proteases or empty vector (pcDNA) were transiently cotransfected into 293T cells. The cells were then treated with PBS or trypsin, and HA cleavage was detected by Western blot analysis of cell lysates using a monoclonal antibody specific for HA. Detection of ß-actin served as loading control. (B) Lentiviral reporter viruses bearing 1918 HA and NA or the VSV-G glycoproteins were generated in 293T cells coexpressing the indicated proteases or empty vector (pcDNA), treated with PBS (black bars) or trypsin (white bars), and used for infection of 293T target cells. Viruses harboring no glycoprotein were generated in parallel as control. Luciferase activities in the cell lysates were determined at 72 h post infection. The results of a representative experiment performed in triplicates are shown. Error bars indicate standard deviation (SD). Comparable results were obtained in a separate experiment. (C) To detect SARS-S cleavage in cis, expression plasmids coding for SARS-S and the indicated proteases or empty vector (pcDNA) were transiently cotransfected into 293T cells, which were then treated with trypsin or PBS. Subsequently, S-protein cleavage was detected by Western blot analysis of cell lysates using a serum specific for the S1 subunit of SARS-S. SARS-S cleavage fragments produced by trypsin and TMPRSS2 are indicated by asterisks. Detection of ß-actin served as a loading control. (D) Effector 293T cells were cotransfected with a SARS-S expression plasmid and a plasmid encoding GAL4-VP16 and mixed with target cells cotransfected with a plasmid encoding a GAL4-VP16 responsive luciferase expression cassette and an ACE2 expression plasmid or protease expression plasmid or empty plasmid. The effector and target cells were mixed, treated with PBS (black bars) or trypsin (white bars) and the luciferase activities in cell lysates quantified at 48 h after cell mixing. The results of a representative experiment performed in triplicates are shown. Error bars indicate standard deviation (SD). Similar results were observed in two independent experiments.

Figure 2. Pulmonary and respiratory sinus expression of SARS-CoV and influenza virus activating proteases and receptors.

Lung (A–D) and sinus (E–H) tissue immunostained for TMPRSS2 (A&E), ACE2 (B&F) and HAT (C&G), or stained for 2,6-linked sialic acid (D&H; detected with elderberry (Sambucus nigra) lectin). All positive reactions are detected with the peroxidase technique (brown) and the tissue is counterstained with haematoxylin (blue). (A) There is strong positive anti-TMPRSS2 immunostaining of bronchial epithelium (lining the bronchus, marked Br), type 2 pneumocytes (P2) and alveolar macrophages (Mp). (B) There is moderately strong positive anti-ACE2 immunostaining of bronchial epithelium (lining the bronchus, marked Br), type 2 pneumocytes (P2) and alveolar macrophages (Mp). (C) There is moderately positive anti-HAT immunostaining of bronchial epithelium (lining the bronchus, marked Br) and alveolar macrophages (Mp), with weakly positive immunostaining of some type 1 (P1) and type 2 pneumocytes (P2). (D) All structures are strongly stained for 2,6-sialic acid except for smooth muscle (SM). (E) There is strong positive anti-TMPRSS2 immunostaining of sinus epithelium (Ep) and lymphoid cells (Ly). (F) There is strong positive anti-ACE2 immunostaining of sinus epithelium (Ep) and lymphoid cells (Ly). (G) There is moderately strong anti-HAT immunostaining of sinus epithelium (Ep) and occasional weakly positive immunostaining of lymphoid cells (Ly). (H) All structures are strongly stained for 2,6-sialic acid. Scale bar = 50 microns (shown in panels D and H and also pertaining to 3 preceding panels in each case).

Figure 3. Tonsil and buccal mucosal expression of SARS-CoV and influenza virus activating proteases and receptors.

Tonsil (A–D) and buccal mucosa (E–H) immunostained for TMPRSS2 (A&E), ACE2 (B&F) and HAT (C&G), or stained for 2,6-linked sialic acid (D&H; detected with elderberry (Sambucus nigra) lectin). All positive reactions are detected with the peroxidase technique (brown) and the tissue is counterstained with haematoxylin (blue). (A) There is strong positive anti-TMPRSS2 immunostaining of tonsillar epithelium (Ep) and lymphocytes (Ly). (B) There is weakly positive anti-ACE2 immunostaining of tonsillar epithelium (Ep), but little obvious positive immunostaining of lymphocytes (Ly). (C) There is strongly positive anti-HAT immunostaining of the basal and superficial, but not the middle, layers of tonsillar epithelium (Ep), but little obvious positive immunostaining of lymphocytes (Ly). (D) All structures are strongly stained for 2,6-sialic acid except for a few areas of cells within the tonsillar epithelium (Ep). (E) There is strong positive anti-TMPRSS2 immunostaining of buccal epithelium (Ep) and of a blood vessel (BV) in the underlying connective tissue (CT). (F) There is strong positive anti-ACE2 immunostaining of buccal epithelium (Ep) and weaker positive immunostaining of a blood vessel (BV) in the underlying connective tissue (CT). (G) There is strong positive anti-HAT immunostaining of buccal epithelium (Ep), but a blood vessel (BV) in the underlying connective tissue (CT) appears negative. (H) All structures are strongly stained for 2,6-sialic acid. Scale bar = 50 microns (shown in panels D and H and also pertaining to 3 preceding panels in each case).

Figure 4. Ileal & myocardial expression of SARS-CoV and influenza virus activating proteases and receptors.

Ileum (A–D) and myocardium (E–H) immunostained for TMPRSS2 (A&E), ACE2 (B&F) and HAT (C&G), or stained for 2,6-linked sialic acid (D&H; detected with elderberry (Sambucus nigra) lectin). All positive reactions are detected with the peroxidase technique (brown) and the tissue is counterstained with haematoxylin (blue). (A) There is strong positive anti-TMPRSS2 immunostaining of ileal epithelium (Ep) and also of lymphocytes (Ly) within the core of the villus. (B) There is strong positive anti-ACE2 immunostaining of ileal epithelium (Ep) and also of lymphocytes (Ly) within the core of the villus. (C) There is strongly positive anti-HAT immunostaining of the basal part of the ileal epithelial cells (Ep), but only weak positive immunostaining of occasional lymphocytes (Ly) within the villus core. (D) All structures are strongly stained for 2,6-sialic acid, including ileal epithelium (Ep) and lymphocytes (Ly). (E) There is strong positive anti-TMPRSS2 immunostaining of cardiac myocytes. (F) There is strong positive anti-ACE2 immunostaining of some cardiac myocytes. (G) There is no anti-HAT immunostaining of cardiac myocytes. (H) There is strong 2,6-sialic acid staining of cardiac myocytes. Scale bar = 50 microns (shown in panels D and H and also pertaining to 3 preceding panels in each case).