【病毒外文文献】2012 Influenza and SARS-Coronavirus Activating Proteases TMPRSS2 and HAT Are Expressed at Multiple Sites in Human Respir

Influenza and SARS Coronavirus Activating Proteases TMPRSS2 and HAT Are Expressed at Multiple Sites in Human Respiratory and Gastrointestinal Tracts Stephanie Bertram 1 Adeline Heurich 1 Hayley Lavender 2 Stefanie Gierer 1 Simon Danisch 3 Paula Perin 3 Jared M Lucas 4 Peter S Nelson 4 Stefan Po hlmann 1 Elizabeth J Soilleux 5 1German Primate Center Go ttingen Germany 2Oxfabs Nuffield Department of Clinical Laboratory Sciences John Radcliffe Hospital University of Oxford Oxford United Kingdom 3Institute of Virology Hannover Medical School Hannover Germany 4Division of Human Biology Fred Hutchinson Cancer Research Center Seattle Washington United States of America 5Department of Cellular Pathology and Nuffield Department of Clinical Laboratory Sciences University of Oxford John Radcliffe Hospital Oxford United Kingdom 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 Citation Bertram S Heurich A Lavender H Gierer S Danisch S et al 2012 Influenza and SARS Coronavirus Activating Proteases TMPRSS2 and HAT Are Expressed at Multiple Sites in Human Respiratory and Gastrointestinal Tracts PLoS ONE 7 4 e35876 doi 10 1371 journal pone 0035876 Editor Volker Thiel Kantonal Hospital St Gallen Switzerland Received November 22 2011 Accepted March 23 2012 Published April 30 2012 Copyright C223 2012 Bertram et al This is an open access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited Funding HL is funded by the NIHR Biomedical Research Centre Programme SB AH SG and SP were supported by the German Federal Ministry of Education and Research BMBF via the project Ecology and Pathogenesis of SARS an Archetypical Zoonosis project codes 01KIO701 01KIO703 PSN was supported by National Cancer Institute NCI grant P01CA085859 The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript Competing Interests Professor Stefan Po hlmann is an academic editor for PLoS ONE This does not alter the authors9 adherence to all the PLoS ONE policies on sharing data and materials The remaining authors have declared that no competing interests exist E mail Elizabeth Soilleux ndcls ox ac uk EJS s poehlmann dpz eu SP These authors contributed equally to this work Introduction Influenza viruses and the SARS coronavirus SARS CoV are highly transmissible respiratory viruses which pose a serious threat to human health The yearly recurring influenza epidemics are associated with significant morbidity and mortality particularly among the elderly and the global spread of pandemic influenza viruses can cause millions of deaths 1 The severe acute respiratory syndrome coronavirus SARS CoV which causes a novel lung disease SARS emerged in 2002 and spread to 26 countries in 2003 with 774 fatal infections 2 Both SARS CoV and influenza viruses circulate in animal reservoirs water fowl influenza and bats SARS CoV 3 4 Therefore the identifica tion of cellular factors essential for viral spread in animal and human cells should allow novel approaches to prevention and therapy The SARS CoV spike protein SARS S and the influenza virus hemagglutinin HA are inserted into the viral membranes and mediate host cell entry For this SARS S and influenza HA bind to host cell receptors ACE2 SARS CoV 5 and 2 6 linked sialic acid on membrane proteins or lipids human influenza viruses 6 and mediate the fusion of the viral membrane with a host cell membrane As a consequence viral components are released into the host cell and can subvert the synthetic capabilities of the host cell for production and release of progeny particles The influenza HA and the SARS S protein are both synthe sized as inactive precursors which transit into their active forms upon cleavage by host cell proteases Cleavage of SARS S and influenza HA is essential for viral infectivity and the responsible proteases are targets for antiviral intervention 7 8 but their nature is incompletely defined Recent evidence indicates that the type II transmembrane serine proteases TTSPs TMPRSS2 TMPRSS4 and HAT can activate human influenza viruses for spread in protease transfected cells 4 9 10 In addition endogenous TMPRSS2 was shown to promote influenza virus spread in the cell lines Caco 2 and Calu 3 11 12 The SARS CoV was found to be activated by cathepsin L upon viral uptake into host cell endosomes 8 However several recent reports demonstrated that expression of TMPRSS2 in target cells rendered cathepsin activity dispensable for infectious entry of PLoS ONE www plosone org 1 April 2012 Volume 7 Issue 4 e35876 SARS CoV 13 15 suggesting that both SARS CoV and influenza viruses can exploit TTSPs to promote their spread Despite the intriguing findings made in cell culture the role of TMPRSS2 and HAT in influenza virus and SARS CoV spread and pathogenesis remains to be defined For this it is essential to determine the extent of TMPRSS2 and HAT expression in viral target cells in human tissues Here we show that TMPRSS2 and HAT are coexpressed with ACE2 and 2 6 linked sialic acids the key receptor determinants of SARS CoV and influenza virus respectively in major portions of the human respiratory tract indicating that these proteases could support SARS CoV and influenza virus spread in humans In addition we demonstrate that HA activation is conserved between human TMPRSS2 and TMPRSS2 of animal species critically involved in zoonotic transmission of influenza virus underlining a potentially important role of this protease in the influenza virus zoonosis Materials and Methods Cell culture 293T cells were obtained from the American Type Culture Collection ATCC and were propagated in Dulbecco s modified Eagle s medium DMEM supplemented with 10 fetal bovine serum FBS penicillin and streptomycin and grown in a humidified atmosphere of 5 CO 2 Cell cell fusion assay For analysis of cell cell fusion 293T effector cells seeded in 6 well plates were CaPO 4 transfected with either empty pcDNA plasmids or plasmids encoding SARS S in combination with plasmid pGAL4 VP16 encoding the Herpes Simplex transactiva tor VP16 fused to GAL4 as described 13 In parallel 293T target cells were seeded in 48 well plates and transfected with plasmids encoding the indicated proteases or empty plasmid together with plasmid pGal5 luc which encodes a promoter with five Gal4 binding sites in front of a luciferase gene Transfected effector and target cells were mixed incubated with trypsin or PBS and fusion was quantified by determination of luciferase activities in cell lysates 48 h after cocultivation using a commercially available kit Promega Madison USA Production of lentiviral pseudotypes and infection experiments For generation of lentiviral pseudoparticles CaPO 4 transfec tions were performed as described 13 Briefly 293T cells were transiently cotransfected with pNL4 3 E R Luc 16 and expression plasmids coding for influenza virus HA and neuramin idase NA or vesicular stomatitis virus glycoprotein VSV G 10 For analysis of HA activation by TTSPs expression plasmids for the indicated proteases 11 13 or empty vector were cotransfected into cells producing pseudoparticles The culture medium was replaced at 16 h and harvested at 48 h post transfection The supernatants were passed through 0 45 mm filters and stored at 280uC For infection pseudoparticles were treated with either PBS or trypsin followed by incubation with 293T target cells for three days before cells were lysed and luciferase activities determined using a commercially available kit Promega Madison USA Analysis of SARS S and 1918 HA cleavage For the detection of HA and SARS S cleavage in cis 293T cells were cotransfected with plasmids encoding SARS S 17 or 1918 HA and plasmids encoding the indicated proteases or empty vector pcDNA For analysis of SARS S cleavage in trans plasmids encoding SARS S 17 and proteases were transfected separately into 293T cells followed by mixing of the transfected cells Subsequently the cells were treated with PBS or trypsin lysed separated via 12 5 SDS PAGE and transferred onto nitrocellulose membranes SARS S was detected by staining with rabbit serum raised against the S1 subunit of SARS S subunit 18 For detection of HA a mouse monoclonal antibody was used 19 As a loading control the stripped membranes were incubated with an anti actin antibody Sigma Deisenhofen Germany Bound antibodies were detected with HRP coupled secondary antibodies Dianova Hamburg Germany Immunostaining of tissue sections Formalin fixed paraffin embedded tissue samples of a wide range of tissues from the respiratory and gastrointestinal tracts as well as the myocardium were obtained from the Oxford Radcliffe Biobank with full ethical approval from the National Research and Ethics Service Oxfordshire Research and Ethics Committee A reference 04 Q1604 21 While all patients gave generic consent for the use of their tissue in research at the time of signing a consent form for surgery informed consent from each patient for the use of tissue in this study was not required by the National Research and Ethics Service because all tissue was anonymised Tissue sections were immunostained for TMPRSS2 HAT and ACE2 or with the elderberry lectin Sambucus nigra that detects 2 6 linked sialic acids Antigen retrieval was performed by pressure cooking in different antigen retrieval solutions Slides were mounted in Aquatex mounting medium Merck UK ACE2 immunostaining affinity purified goat polyclonal serum R 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 epitheli um 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 doi 10 1371 journal pone 0035876 g002 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 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 lympho cytes 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 immunostain ing 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 doi 10 1371 journal pone 0035876 g003 Proteolytic Activation of Influenza and SARS PLoS ONE www plosone org 5 April 2012 Volume 7 Issue 4 e35876 since TMPRSS2 was absent from this cell type and expression of HAT was infrequent and weak However other studies found that type II pneumocytes are preferentially infected 41 and these cells were identified as positive for 2 6 linked sialic acid TMPRSS2 and occasionally for HAT within the present study The presence of cells positive for 2 6 linked sialic acid TMPRSS2 and or HAT was not limited to the respiratory tract in keeping with published findings which demonstrate TMPRSS2 expression in the epithelia of several organs 21 42 45 It is therefore tempting to speculate that TMPRSS2 and HAT might also support viral spread outside the lung and might thus contribute to complications associated with influenza infection like gastrointestinal manifestations myocarditis and encephalopathy 38 In sum TMPRSS2 and with the exception of the alveolar epithelium HAT could activate influenza viruses throughout the respiratory tract and might support viral spread in extra respiratory tissues The mode of cleavage activation is a major virulence determinant of avian influenza viruses 11 46 48 Viruses with a multi basic cleavage site in HA are believed to be activated by ubiquitously expressed host cell proteases and can thus replicate systemically and cause severe disease 11 In contrast it has been posited that replication of viruses with a mono basic cleavage site is confined to the aerodigestive tract because the expression of as yet unidentified HA activating protease s is limited to this organ 11 Our results suggest that TMPRSS2 could be the elusive protease but it remains to be demonstrated whether TMPRSS2 expression in waterfowl and poultry is indeed specific for the aerodigestive tract HA activation was conserved between avian porcine and human TMPRSS2 which share high sequence identity see figure S2 indicating that TMPRSS2 might support influenza virus spread not only in the reservoir waterfowl and humans but also in an important intermediate host swine The SARS CoV causes a severe respiratory illness with fatal outcome in about 10 of the afflicted individuals The metalloprotease ACE2 has been identified as the SARS CoV receptor and expression of ACE2 on type II pneumocytes the major viral target cells and other pulmonary cells has been demonstrated 49 52 A cornerstone study by Simmons and colleagues showed that infectious SARS CoV entry into cell lines depends on the activity of endosomal cathepins particularly cathepsin L 8 suggesting that cathepsin activity might be required for viral spread in the respiratory tract However several studies showed that TMPRSS2 activates SARS CoV for cathep sin independent host cell entry and demonstrated TMPRSS2 expression in type II pneumocytes 13 15 In addition a recent report indicates that HAT can promote SARS S driven cell cell but not virus cell fusion 37 Thus the SARS CoV may use TTSPs in addition to or instead of cathepsins to ensure its activation in key target cells Our analysis confirms and extends these findings by demonstrating that ACE2 and TMPRSS2 are coexpressed by cells in the nasal and buccal mucosa as well as in the epithelia of bronchus and larynx Thus one can speculate that in the context of the infected host cathepsin activity might not be essential for SARS CoV spread in most parts of the respiratory tract Experiments with cathepsin inhibitors and knock out mice are required to address this possibility The gastrointestinal tract is a well established target of SARS CoV 49 and it has actually been suggested that a wide range of tissues and organs can be infected by the virus 53 Our finding that TMPRSS2 and ACE2 are coexpressed in colon and various other tissues indicates that also the extrarespiratory spread of SARS CoV might be promoted by TMPRSS2 Collectively our findings are compatible with an important role of TMPRSS2 and HAT in influenza virus and of TMPRSS2 in SARS coronavirus infection Knock down of these proteases in primary pulmonary cells and the analysis of Tmprss2 knock out mice would allow to precise definition of the role of these proteases in viral spread The latter animals are available 54 and do not show an obvious phenotype indicating that TMPRSS2 might be an attractive target for novel drugs active against respiratory viruses Figure 4 Ileal 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 doi 10 1371 journal pone 0035876 g004 Proteolytic Activation of Influenza and SARS PLoS ONE www plosone org 6 April 2012 Volume 7 Issue 4 e35876 Supporting Information Figure S1 To detect SARS S cleavage in trans expres sion plasmids coding for SARS S and the indicated proteases or empty vector pcDNA were separately transfected into 293T cells Subsequently the SARS S and protease expressing cells were mixed treated with trypsin or PBS and S protein cleavage was detected by Western blot analysis 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 actin served as a loading control TIF Figure S2 The amino acid sequences of TMPRSS2 of human NCBI Reference SequenceNP 001128571 1 chicken XM 416737 3 swine BAF76737 1 and mouse origin AAF97867 1 were aligned using VectorNTI Identical amino acids are marked in black similar amin