Activation of influenza A viruses by host proteases from swine airway epithelium

Abstract

Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways.

FIG 1

Characterization of isolated PTECs and PBECs. Monolayers of primary PTECs and PBECs were analyzed by light microscopy (magnification, ×10) and immunofluorescence staining of the epithelial marker protein cytokeratin using cytokeratin-specific antibodies and FITC-conjugated secondary antibodies (magnification, ×20). The adherens junction protein E-cadherin was visualized with anti-E-cadherin antibodies and TRITC-conjugated secondary antibodies (magnification, ×40). Nuclei were counterstained with DAPI.

FIG 2

Proteolytic activation and multicycle replication of influenza A viruses in primary porcine airway epithelial cells. (A) Cultures of primary PTECs and PBECs were infected with human influenza virus isolate A/Hamburg (H1N1)pdm09 or A/Aichi (H3N2) at an MOI of 0.003 to 0.007 and incubated for 24 h to allow multiple cycles of virus replication. Infected cells were immunostained against the viral NP. (B) Infection of PTECs and PBECs with A/Hamburg (H1N1)pdm09 (left) or A/Aichi (H3N2) (right) at a low MOI of 0.001 to 0.0001. At the indicated time points, the virus titers of trypsin-treated or untreated supernatants were determined by plaque assay. The results shown are mean values of three independent experiments. (C) At 72 h p.i., virus-containing supernatants were treated with trypsin or remained untreated and pelleted by ultracentrifugation, and HA cleavage was analyzed by SDS-PAGE under reducing conditions and Western blotting using HA-specific antibodies. M1, influenza virus matrix protein.

FIG 3

Distribution of swTMPRSS2- and swAT-specific mRNA in respiratory tissues and expression of recombinant swine and human proteases. (A) Total RNA was isolated from PEECs, PTECs, and PBECs as well as porcine lung tissue and used as a target for RT-PCR analysis with a set of primers specific for swTMPRSS2, swAT, or α-tubulin. H₂O was used as a control. (B) 293T cells were transiently transfected with either empty pCAGGS plasmid (mock) or pCAGGS encoding either swTMPRSS2-FLAG, swAT-FLAG, TMPRSS2-FLAG, or HAT-FLAG. At 24 h p.t., cell lysates were subjected to SDS-PAGE under reducing conditions, subsequently transferred to a PVDF membrane, and analyzed by use of FLAG-specific antibodies and HRP-conjugated secondary antibodies. Open and filled arrowheads indicate the zymogen and the mature form of each protease, respectively.

FIG 4

Proteolytic activation of influenza virus HA by swTMPRSS2 and swAT. (A) 293T cells were cotransfected with plasmids encoding HA of A/Hamburg (H1N1)pdm09 and either pCAGGS (mock), pCAGGS-swTMPRSS2, or pCAGGS-swAT. At 48 h p.t., HA cleavage was detected by Western blot analysis of cell lysates using HA-specific antibodies. Treatment of HA-expressing cells with trypsin served as a control. *, a nonspecific band. (B) At 8 h after transfection with empty vector pCAGGS (without [w/o] protease) or protease-encoding plasmids, MDCK cells were infected with influenza virus A/Hamburg (H1N1)pdm09 or A/Aichi (H3N2) at an MOI of 0.01 to 0.001 for 24 h. Infected cells were immunostained against NP. Magnification, ×10.

FIG 5

Localization of swTMPRSS2 and swAT in mammalian cells transiently expressing protease and primary PTECs and PBECs. (A) MDCK cells were transfected with either pCAGGS-swTMPRSS2-FLAG or pCAGGS-swAT-FLAG. At 24 h p.t., cell surface expression of swTMPRSS2 and swAT was analyzed using FLAG-specific antibodies and FITC-conjugated secondary antibodies. (B) MDCK cells transiently expressing swTMPRSS2 and swAT were fixed and permeabilized, and the intracellular localization of each protease was determined by indirect immunofluorescence using FLAG-specific antibodies and FITC-labeled secondary antibodies. Nuclei were counterstained with DAPI. (C) swTMPRSS2 and swAT transiently coexpressed in permeabilized Huh7 cells were visualized by polyclonal FLAG and HAT antisera, respectively, and fluorescence-labeled secondary antibodies. (D) Huh7 cells transiently expressing swTMPRSS2-FLAG were fixed, permeabilized, and analyzed for the localization of swTMPRSS2 and endogenous TGN38 using FLAG- and TGN38-specific primary antibodies and species-specific FITC- or TRITC-conjugated secondary antibodies. (E, F) Huh7 cells were cotransfected with plasmids encoding the TGN-residing protease furin and either swTMPRSS2-FLAG or swAT-FLAG for 24 h. The subcellular localization of the proteases was analyzed by FLAG- or furin-specific antibodies and species-specific fluorescence-conjugated secondary antibodies. (G) Cell surface expression of endogenous swTMPRSS2 and swAT in primary PTECs and PBECs was detected with polyclonal TMPRSS2 and HAT antisera and FITC-labeled secondary antibodies. (H) Monolayers of primary PTECs and PBECs were fixed and permeabilized, and endogenous swTMPRSS2 and swAT were visualized with anti-TMPRSS2 or anti-HAT serum, respectively, and FITC-conjugated antibodies.

FIG 6

Enzymatic activity of swTMPRSS2 and swAT at the cell surface and proteolytic activation of incoming virions. (A) At 24 h after transfection of MDCK cells with either empty vector pCAGGS (mock) or expression plasmids encoding swTMPRSS2, swAT, or HAT as the control, the cells were incubated with the fluorogenic peptide substrate Boc-Gly-Pro-Arg-AMC. Protease activity at the cell surface was measured by determination of the relative fluorescence units (RFU) of the released AMC at the indicated time points. The results are the mean enzymatic activities for three independent experiments, with values for mock-transfected cells subtracted from values for protease-expressing cells within each experiment. (B, C) MDCK cells transiently expressing swTMPRSS2, swAT, and HAT (B) and primary PTEC and PBEC monolayers (C) were inoculated with human influenza virus isolate A/Memphis (H1N1) containing noncleaved HA0 (∼10³ virions per well) and subsequently incubated for 10 h at 37°C to allow a single cycle of replication. Additional inoculation with virions in the presence of aprotinin (Apr) served as a control. Cells were fixed and immunostained against NP, and the number of infected cells per well was determined. The results are the mean values of a representative experiment, with each sample measured in triplicate.