Matriptase, HAT, and TMPRSS2 activate the hemagglutinin of H9N2 influenza A viruses

Abstract

Influenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.

Fig 1

Structures of the matriptase (MI-021 and MI-462) and furin (MI-701) inhibitors used.

Fig 2

Proteolytic activation of H9N2 viruses with mono-, di-, and tribasic cleavage sites by TMPRSS2 and HAT. (A) MDCK-HAT and MDCK-TMPRSS2 cells with doxycycline-induced expression (+) of either HAT or TMPRSS2 were infected with H9N2 isolate H9-Wisc, H9-782, or H9-2061 at an MOI of 0.1. Cells infected in the absence of doxycycline (−) were used as a control. At 24 h p.i., virus-containing supernatants were analyzed by SDS-PAGE and Western blotting using a polyclonal H9N2 serum that recognizes HA0 and HA1. The amino acid sequence at the HA cleavage site of each virus is indicated in the one-letter code. (B) MDCK-HAT and MDCK-TMPRSS2 cells were infected with the indicated H9N2 viruses at an MOI of 0.0001 and incubated in the presence (+) or absence (−) of doxycycline. At indicated time points p.i., viral titers were determined by plaque assay. Results are the mean values from three independent experiments.

Fig 3

Matriptase activates H9 with R-S-S-R and R-S-R-R cleavage sites. (A) 293T cells were transfected with plasmids encoding the HA of H9-782, H9-2061, H9-2061mut, or H9-Wisc. At 24 h posttransfection, cells were harvested and either treated with soluble matriptase (M) or trypsin (T) or remained untreated (∅). Cells were lysed and subjected to SDS-PAGE under reducing conditions and Western blot analysis using antibodies against H9N2. The additional band in H9-Wisc-expressing cells represents nonglycosylated HA0 (HA0*). The amino acid sequence at the HA cleavage site of each virus is indicated in the one-letter code. (B) MDCK(H) cells were infected with H9-Wisc, H9-782, H9-2061, or H9-2061mut at an MOI of 0.0001 and incubated in the presence of soluble matriptase (3 μg/ml) for 48 h. At 16, 24, and 48 h p.i., virus propagation was analyzed by plaque titration. The results shown are the mean values from two independent experiments.

Fig 4

Proteolytic activation of H9-782 and H9-2061 in MDCK-II cells and its suppression by matriptase inhibitors. (A) MDCK(H) and MDCK-II cells were infected with either H9-782, H9-2061, or H9-Wisc and incubated with an Avicel overlay with (+) or without (−) trypsin for 48 h. Infected cells were fixed and immunostained using antibodies against H9N2. (B) MDCK-II cells were infected with H9N2 virus H9-782 or H9-2061 at a low MOI and incubated in the presence of matriptase inhibitor MI-021 or MI-462 (each at 50 μM) for 48 h. At the indicated time points, virus titers were determined by plaque assay. w/o, without. The growth curves shown are the mean values from three independent experiments.

Fig 5

Proteolytic activation of H9-2061 and H9-782 in chicken embryo kidney (CEK) epithelial cells. (A) Primary CEK cells were infected with the indicated viruses at an MOI of 0.01 to 0.001 and incubated in the absence (−T) or presence (+T) of trypsin (0.5 μg/ml) for 24 h. Cells were fixed and immunostained using an antibody against NP and an FITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI. (B) Primary CEK cells were infected with the indicated virus and incubated in either the absence or presence of trypsin (0.5 μg/ml) and with or without matriptase inhibitor MI-462 (50 μM). At 24 h p.i., cell lysates were subjected to SDS-PAGE and immunoblotting using antibodies against H9N2. The amino acid sequences of the HA cleavage sites are indicated in the one-letter code. (C) As controls, CEK cells were infected with HPAIV H7-Italy and LPAIV H10-Germany in the absence or presence of trypsin for 24 h and immunostained against NP as described. (D) CEK cells were infected with the indicated virus at an MOI of 0.01 and incubated for 24 h with or without trypsin and in the presence or absence of peptide-mimetic inhibitors of matriptase (MI-462) or furin (MI-701) (each at 50 μM). Cell lysates were analyzed by SDS-PAGE and immunoblotting using virus-specific antibodies. The amino acid sequence at the HA cleavage site of each virus is indicated in the one-letter code.

Fig 6

HA of human H1 and H3 viruses is not cleaved by matriptase. 293T cells were transfected with pCAGGS plasmids encoding the HA of pandemic viruses H1-SC18, H3-HK68, and H1-HH09 (Table 1) or the HA cleavage site mutant H1-HH09mut. The amino acid sequence at the cleavage site of each HA is indicated in the one-letter code. At 24 h posttransfection (p.t.), cells were incubated with soluble matriptase (M) or trypsin (T) or were mock treated (∅), and subsequently cell lysates were subjected to SDS-PAGE and Western blotting using H1- or H3-specific antibodies.