Kallikrein 13: a new player in coronaviral infections

Aleksandra Milewska; Katherine Falkowski; Magdalena Kalinska; Ewa Bielecka; Antonina Naskalska; Pawel Mak; Adam Lesner; Marek Ochman; Maciej Urlik; Jan Potempa; Tomasz Kantyka; Krzysztof Pyrc

HCoV-HKU1 replication in the presence of both inhibitors (Fig. 2B) . All inhibitors were used 151 at non-toxic concentrations (Fig. 2C) . 152 The experiments conducted so far suggested that KLK13 is required for virus infection. 153 However, one may question the specificity of the KLK13 protease inhibitors. To ensure that 154 KLK13 is indeed the priming enzyme during HCoV-HKU1 infection, we developed HAE 155 cultures by transforming cells with lentiviral vectors encoding shRNAs targeting KLK13 156 mRNA. We then confirmed that the expression of the protease was silenced (HAE_shKLK13). (Fig. 3A) . Importantly, HAE_shKLK13 cells continued to differentiate and formed 162 pseudostratified cultures (Fig. 3B) . Next, we infected HAE_ctrl, HAE_GFP, HAE_vector, and 163 HAE_shKLK13 with HCoV-HKU1 (10 6 RNA copies per ml) and incubated them for 2 h at 164 32°C with the viral stock solution. Cultures were maintained at 32°C for 5 days at an air-liquid 165 interface. Apical washes were collected, and virus yield was determined by RT-qPCR. We 166 found that, in contrast to that in control cultures, replication of virus in HAE_shKLK13 was 167 abolished (Fig. 3C) . Overall, these data suggest that silencing the KLK13 gene in HAE inhibits 168 virus infection, indicating that KLK13 is necessary for HCoV-HKU1 infection. 171 We determined that KLK13 is essential for efficient HCoV-HKU1 infection in HAE The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.01.971499 doi: bioRxiv preprint proteases my render RD cells permissive, we generated RD cells expressing human KLK13 or 176 TMPRSS2 proteases. RD cells were transduced with lentiviral vectors harboring the KLK13 177 gene (RD_KLK13), control vector (RD_ctrl), or TMPRSS2 (RD_TMPRSS2). Due to the lack 178 of KLK13 specific antibodies, we verified its presence based on RT-PCR (Fig. 4A) . The 179 presence of TMPRSS2 in RD_TMPRSS2 cells was confirmed using Western blot. The 180 TMPRSS2 band in RD cells was observed at 25 kDa, which corresponds to one of the naturally 181 occurring splicing variants (Fig. 4B) . Subsequently, we transduced RD_ctrl, RD_KLK13 and pseudoviruses, compared to ΔEnv, which was completely abolished in the presence of KLK13 193 inhibitor (Fig 4D) . Overall, these data demonstrated that KLK13 activity drives HCoV-HKU1 194 entry into cells. 195 196 KLK13 enables the replication of HCoV-HKU1 in RD cells 197 Obtained results showed that KLK13 expression on RD cells was sufficient for HCoV-198 HKU1 pseudovirus entry. Here, we aimed to test whether KLK13 presence renders RD cells 199 permissive for HCoV-HKU1 infection. For this, we infected RD_ctrl and RD_KLK13 cells 200 author/funder. All rights reserved. No reuse allowed without permission.