Human matriptase/ST 14 proteolytically cleaves H7N9 hemagglutinin and facilitates the activation of influenza A/Shanghai/2/2013 virus in cell culture

Abstract Background Influenza is a zoonotic disease that infects millions of people each year resulting in hundreds of thousands of deaths, and in turn devastating pandemics. Influenza is caused by influenza viruses, including influenza A virus (IAV). There are many subtypes of IAV but only a few seem to be able to adapt to humans and to cause disease. In 2013, an H7N9 IAV subtype emerged in China that does not cause clinical symptoms in its chicken host but leads to severe infections when transmitted into humans. Since 2013, there have been six epidemic waves of H7N9 with 1567 laboratory‐confirmed human infections and 615 deaths. Pathogenicity of IAV is complex, but a crucial feature contributing to virulence is the activation of the hemagglutinin (HA) fusion protein by host proteases that triggers membrane fusion and leads to subsequent virus propagation. Methods 293T, VERO, and MDCK cells were used to conduct Western blot analysis, immunofluorescence assays, and pseudoparticle and live virus infections, and to evaluate H7N9 HA cleavage‐activation. Results/Conclusions We show that human matriptase/ST 14 is able to cleave H7N9 HA. Cleavage of H7N9 HA expressed in cell culture results in fusogenic HA and syncytia formation. In infection studies with viral pseudoparticles carrying matriptase/ST 14‐activated H7N9 HA, we observed a high infectivity of cells. Finally, human matriptase/ST 14 also activated H7N9 live virus which resulted in high infectivity. Our data demonstrate that human matriptase/ST 14 is a likely candidate protease to promote H7N9 infections in humans.


| INTRODUC TI ON
Influenza A viruses (IAVs) circulate in waterfowl as their natural reservoir and are categorized based on the antigenic properties of their hemagglutinin (HA) and neuraminidase (NA) proteins. There are 16 different HAs and nine different NA present in wild birds. 1 Frequently, wild birds infect poultry resulting in the culling of millions of birds and significant economic losses. However, wild birds rarely show disease symptoms while IAV in poultry usually exhibits respiratory tropism and is able to mutate into a more highly pathogenic form. 2 Influenza is also a zoonotic disease, and the virus is occasionally transmitted from infected poultry to humans. Certain HA/ NA combinations, however, seem to be more favorable for human infections. H1N1, H2N2, and H3N2 have caused several devastating outbreaks in humans in the past such as the 1918 "Spanish flu," the 1957 "Asian pandemic," the 1968 "Hong Kong pandemic," and the 2009 "Swine flu," respectively. 3 Currently, the recently emerged H7N9 strain is of concern for global public health. Low-pathogenic avian influenza (LPAI) H7N9 was first discovered in humans in 2013 in China when three patients were hospitalized with severe and fatal influenza. 4 Since then, there have been six epidemic H7N9 waves that resulted in 1567 laboratory-confirmed human infections and 615 deaths and a seventh wave is currently ongoing. 5 Intriguingly, the virus isolated from the 2013 outbreak was suggested to be derived from chickens that did not show any (or only very mild) symptoms of disease. Further investigations revealed that the viral genes were exclusively from avian origin. 4 To this date, there are no reports of H7N9 human to human transmissions even though it cannot be excluded in very few cases.
However, most cases of human H7N9 infections were attributed to exposure of humans to infected wild birds or poultry and live animal markets. 5 A crucial step in the life cycle of H7N9 (and all other influenza subtypes) is the cleavage of its HA fusion protein by host proteases.
HA is synthesized as a precursor and requires proteolytic processing to exert its fusogenic activity. Cleavage occurs at the C-terminus of the fusion peptide and results in its exposure allowing HA to induce fusion of the viral and cellular membrane and subsequent release of the RNA into the host cell. 6,7 LPAI viruses have a monobasic cleavage site that consists of a single arginine, which is preceded by three amino acids that are influenza subtype-specific. While LPAI viruses in wild birds are usually cleaved by trypsin in their gastro-intestinal tract, there are trypsin-like serine proteases in humans and poultry that reside in the respiratory tract and which are able to process these viruses. Therefore, the disease is usually confined to this tissue. 8 Only very little is known about the activation of H7N9 viruses. Previously, it was reported that the type II transmembrane serine protease TMPRSS2 plays a role in the cleavage of H7N9 HA and activation of the virus in mice. 9,10 Both studies showed that TMPRSS2 knockout (KO) mice were highly tolerant to H7N9 infections. When KO mice were complemented with human or mouse TMPRSS2, their susceptibility to the virus was restored. 9 We recently reported that human matriptase/ST 14 was able to very efficiently cleave a H7N9 cleavage motif peptide mimic. 11 Matriptase/ST 14 is a membrane-bound protease localized in epithelial cells of various tissues in humans that seems to play a significant role in the activation of various influenza A subtypes such H1N1 and H9N2. 12,13 However, results obtained using cleavage site peptide mimics do not always reflect the in vivo situation and need to be validated. 11,14 Here, we show that human matriptase/ ST   The pCMV-MLVgag-pol murine leukemia virus (MLV) packaging construct, the pTG-Luc transfer vector encoding luciferase reporter, and pCAGGS/VSV-G plasmid were described before. 15,16 Recombinant human matriptase/ST 14 was purchased from R&D systems. The specific activity of the enzyme is described as 10 000 pmol/min/µg for the fluorogenic peptide substrate Boc-QAR-AMC. 150 mM NaCl, pH 7.4) was added to one well and incubated on a rocker at 37°C and 5% CO 2 for 90 minutes. For the control, 0.8 mM Trypsin in PBS was added for 10 minutes. Cells were then processed for Western blot analysis as previously described. 12 The antibody to detect A/Shanghai/2/2013 H7N9 HA (NR48765) was obtained from the Biodefense and Emerging Infections Research Resources Repository. The secondary antibody had an Alexa fluor 488 tag and was purchased from Invitrogen. Western blot membranes were scanned using a ChemiDoc imaging system (Bio-Rad) to facilitate exposure in a linear range. Bands were quantified using ImageJ software, and cleavage efficiencies for HA 1 were calculated by the following equation: (HA 1 /[HA 0 + HA 1 ])*100. Graph was generated using Graphpad Prism 7 software.

| Pseudoparticle production and infection
For pseudoparticle production, 293T cells were grown in 6-well plates and transfected with pDZ/H7N9 HA, pDZ/H7N9 NA, pCMV-MLVgag-pol, and pTG-Luc using Turbofect according to the manufacturer's recommendations. Controls were transfected with pCAGGS/VSV-G, pCMV-MLVgag-pol, and pTG-Luc (positive control) or only pCMV-MLVgag-pol and pTG-Luc (negative control). Supernatants were harvested 72 hours post-transfection, and pseudoparticles were stored at −80°C. Infections, cell lysis, and data analysis were performed as previously described. 17 Experiment was repeated three independent times. Results were plotted, and statistical analysis was conducted using Graphpad Prism7 software. immuno-plaque assay as previously described. 18 The experiment was repeated three independent times. Data and statistical analysis were performed using Graphpad Prism7 software.

| H7N9 HA is cleaved by human matriptase/ST 14 and results in a fusogenic active protein
We previously reported that recombinant human matriptase/ In conclusion, recombinant human matriptase/ST 14 proteolytically processes H7N9 HA that is then able to exert its fusogenic activity.

| Viral entry is facilitated by matriptase/ST 14 cleaved H7N9 HA
After we established that recombinant human matriptase/ST 14-mediated cleavage of results in a fusogenic H7N9 HA protein, we wanted to test whether it would promote the infectivity of the virus. As a first step, we chose to produce pseudoparticles representing surrogates of native virions that allowed us to study the viral entry into host cells. The pseudoparticles used in this assay possess a MLV core that incorporates a luciferase reporter gene upon their release from the producing cells. 17 To facilitate fusion, the pseudovirions contained H7N9 HA and were incubated with trypsin or matriptase/ST 14 prior to their addition to cells. We Pseudoparticles carrying VSV-G were highly infective showing that the pseudoparticle production worked as expected ( Figure 3A, Table 1A). H7N9 HA pseudovirions treated with trypsin and with matriptase/ST 14 were significantly more infectious compared to the same pseudoparticles without protease treatment ( Figure 3B, Table 1A). However, there was no significant difference between trypsin-and matriptase/ST 14-treated pseudoparticles suggesting that all pseudovirions were depleted ( Figure 1B, Table 1A).
Pseudoparticles without an incorporated fusion protein were not infectious. The data show that matriptase/ST 14-mediated cleavage of H7N9 HA results in fully infectious virions that are able to fuse with host cells.

| Matriptase/ST 14 promotes viral growth of A/ Shanghai/2/2013 H7N9
Next, we investigated whether matriptase/ST 14 was able to sustain and promote viral growth of A/Shanghai/2/2013 H7N9 in a cell culture system. Therefore, we infected MDCK cells with the live virus at a low MOI of 0.1 and added trypsin or matriptase/ST 14. The control did not receive protease treatment. After 48 hours, the supernatants were collected, and the viral titers were quantified using an immunoplaque assay.