TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells

Abstract

Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.

FIG. 1.

Cleavage of influenza virus hemagglutinin by TMPRSS2, TMPRSS4, or hepsin. (A) Cleavage of hemagglutinin (HA) in 293T cells expressing TMPRSS2, TMPRSS4, or hepsin. The HA of the 1918 influenza virus and the protease indicated were transiently coexpressed in 293T cells. The cells were then treated with trypsin or PBS, and HA cleavage was detected by Western blot analysis of cell lysates. Detection of β-actin served as a loading control. Mock, cells transfected with empty vector alone; pcDNA, cells cotransfected with an HA expression plasmid and empty vector. (B) Incorporation of cleaved HA into virions. HA was expressed as described for panel A; however, cells were additionally cotransfected with plasmids encoding the HIV-1 Gag protein and the 1918 influenza virus NA. VLPs were concentrated from culture supernatants by centrifugation, and HA cleavage and Gag contents were analyzed by Western blotting. Mock, VLPs produced in cells transfected with a Gag expression plasmid and empty vector; pcDNA, VLPs produced in cells cotransfected with plasmids encoding Gag, HA, and NA and empty vector. (C) Expression of TMPRSS2 or hepsin, but not TMPRSS4, alters the glycosylation of HA1. 293T cells, transfected either with expression vectors for the indicated proteases or with empty vector, were used for the production of HA-bearing VLPs as described for panel B, and concentrated particles were subsequently treated with trypsin or PBS. Thereafter, the VLPs were either digested with PNGase F or mock treated, and HA cleavage was analyzed by Western blotting.

FIG. 2.

TMPRSS2 and TMPRSS4, but not hepsin, activate influenza virus hemagglutinin by cleavage. (A) Cleavage activation of lentiviral pseudotypes bearing HA. (Left) Lentiviral reporter viruses bearing 1918 HA and NA were generated in 293T cells coexpressing the indicated protease, treated with PBS or trypsin, and used for the infection of Huh7 target cells. Luciferase activities in cell lysates were determined at 72 h postinfection. The results of a representative experiment performed in triplicate are shown; error bars indicate standard deviations. Comparable results were obtained in two independent experiments. (Right) Incorporation of HA into lentiviral pseudotypes. Lentiviral pseudotypes generated in 293T cells coexpressing the indicated protease were pelleted through a sucrose cushion, and the incorporation of influenza virus HA and HIV-1 capsid protein (p24) into virions was assessed by Western blotting. (B) Cleavage activation of PR8 (H1N1) and SCM35 (H7N7). The indicated proteases were transiently expressed in 293T cells, and the cells were infected at an MOI of 0.01 with PR8 (H1N1) or SCM35 (H7N7) produced in hens' eggs. Subsequently, the cells were treated with PBS or trypsin as indicated, and at 24 h postinfection, viral spread was quantified as the release of infectious particles into the culture supernatants, as measured by a focus formation assay. The results of a representative experiment performed in triplicate are shown; error bars indicate standard deviations. Comparable results were obtained in a separate experiment.

FIG. 3.

TMPRSS2 and TMPRSS4 mRNAs and TMPRSS2 protein are expressed in Caco-2 cells. (A) Analysis of TMPRSS2 and TMPRSS4 mRNA expression by RT-PCR. Total RNA was prepared from the indicated cell lines, treated with DNase, reverse transcribed, and used for the amplification of TMPRSS2, TMPRSS4, and GAPDH mRNAs by nested PCR. The results of single gels from which irrelevant lanes were removed are shown. (B) Analysis of TMPRSS2 and TMPRSS4 mRNA expression by quantitative RT-PCR. RNA was prepared as described for panel A, and TMPRSS2, TMPRSS4, and GUSB (housekeeping control) were amplified by TaqMan gene expression assays. Similar results were obtained in two independent experiments. No TMPRSS4 signal was detected in Vero E6 cells. (C and D) Expression of TMPRSS2 (C) or TMPRSS4 (D) in cell lines as determined by Western blotting. The indicated cell lines were lysed, and the expression of TMPRSS2 or TMPRSS4 was analyzed by Western blotting. Detection of β-actin served as a loading control. The results of a single gel from which irrelevant lanes were removed are shown in panel C. 293T + TMPRSS2, 293T cells transiently expressing TMPRSS2; 293T + TMPRSS4, 293T cells transiently expressing TMPRSS4.

FIG. 4.

Influenza virus HA is activated in Caco-2 cells in the absence of trypsin. (A) Activation of HA pseudotypes in Caco-2 cells. Caco-2 and 293T cells were transiently cotransfected with plasmids encoding a lentiviral vector and 1918 HA and NA. The supernatants were treated with trypsin or PBS and were then used to infect Huh7 target cells. Luciferase activity in cell lysates was determined at 72 h postinfection. The results ± standard deviations of a representative experiment performed in triplicate are shown and were confirmed in three separate experiments. (B) Analysis of HA cleavage in Caco-2 and 293T cells. 293T and Caco-2 cells were transfected with 1918 HA, or 293T cells were cotransfected with 1918 HA and TMPRSS2 expression plasmids, and HA cleavage in cell lysates was detected by Western blotting. The results of a single gel from which irrelevant lanes were removed are shown. (C) Activation of influenza viruses in Caco-2 cells. Caco-2 and 293T cells were first infected with PR8 (H1N1) or SC35M (H7N7) at an MOI of 0.01 and then treated with PBS or trypsin. The release of infectious particles in the supernatant was determined by a focus formation assay. Results of a representative experiment performed in triplicate are shown, and activation of PR8 (H1N1) in Caco-2 but not 293T cells was confirmed in two separate experiments. (D) Infection of Caco-2 and 293T cells with pseudotypes bearing nonactivated or trypsin-activated HA. Pseudotypes bearing the 1918 HA, the 1918 NA, or the G protein of VSV were first treated either with PBS or with trypsin and then used to infect 293T and Caco-2 cells. Luciferase activities in cell lysates were determined at 72 h postinfection. Similar results were obtained in two independent experiments. (E) Infection of TTSP-expressing 293T cells with pseudotypes bearing nonactivated or trypsin-activated HA. The indicated TTSPs were transiently expressed in 293T cells, and the cells were infected with HA-bearing pseudotypes as described for panel D.

FIG. 5.

Knockdown of TMPRSS2 and TMPRSS4 inhibits the trypsin-independent spread of influenza virus in Caco-2 cells. (A) Analysis of TMPRSS2 knockdown. Caco-2 cells either were transfected, by use of Lipofectamine, with a nonsense control siRNA or a TMPRSS2- or TMPRSS4-specific siRNA in the presence or absence of the control siRNA or were cotransfected with a 1:1 mixture of TMPRSS2- and TMPRSS4-specific siRNAs. TMPRSS2 expression in transfected cells was analyzed by Western blotting. Detection of β-actin served as a loading control. All siRNA transfection mixtures contained 200 pmol siRNA. Thus, if the transfection mixture contained a single siRNA species, 200 pmol of the siRNA was added. If two different siRNA species were cotransfected, 100 pmol of each was added. (B) Caco-2 cells were first transfected with siRNAs as described for panel A and then infected with PR8 (H1N1) or SC35M (H7N7) at an MOI of 0.01 in the presence or absence of trypsin. The release of infectious particles into the supernatant was determined by a focus formation assay. The results of a representative experiment ± standard deviations are shown and were confirmed by three separate experiments for PR8 (H1N1) and by one separate experiment for SC35M (H7N7). The statistical significance (asterisks) of the inhibitory effects of TMPRSS2- and TMPRSS4-specific siRNAs relative to that of the nonsense control siRNA was calculated by using the two-tailed Student t test for correlated samples.

FIG. 6.

Expression of TMPRSS2 in human alveolar epithelium. (A) Hematoxylin-and-eosin-stained section of a normal lung showing several alveolar spaces, in which alveolar macrophages (M), type I pneumocytes (P1), and type II pneumocytes (P2) are labeled. Bar, 20 μm. (B) A serial section of panel A immunostained for TMPRSS2 using the peroxidase technique (brown) shows strong positive staining in type II pneumocytes and alveolar macrophages. (C) A serial section of panel B stained with Sambucus nigra lectin shows strong positive membrane staining of all cell types, including type II pneumocytes and alveolar macrophages. (D) A serial section of panel C immunostained with an irrelevant mouse primary antibody (Melan-A), as a negative control for panel B, shows no immunostaining. Alveolar macrophages show a faint brown tint, due to the presence of carbon, but not the strong brown staining of macrophages seen in panel B. A serial section of panel D immunostained using a goat polyclonal serum as a primary antibody, as a negative control for panel C, appeared very similar to panel D (data not shown).