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

The copyright holder for this preprint (which was not peer-reviewed) is the .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 with HCoV-HKU1 (10 8 RNA copies per ml) and incubated the culture for 7 days at 32°C in the 201 presence or absence of a KLK13 inhibitor (10 µM) or DMSO. Next, cellular RNA was isolated 202 and the presence of HCoV-HKU1 N subgenomic mRNA (N sg mRNA), which is considered 203 to be a hallmark of coronaviral infection, was assessed (14). sg mRNA appeared in RD_KLK13 204 cells, while no signal was detected in cultures supplemented with the KLK13 inhibitor nor in 205 RD_ctrl cells (Fig. 5A) . 206 To further confirm the role of KLK13 during HCoV-HKU1 infection RD HCoV-HKU1 infection was analyzed by means of N sg mRNA detection. Again, we found that 212 N sg mRNA was produced only in the presence of KLK13 (Fig. 5B) . Further, we passaged in the presence of KLK13 (Fig. 5B) . However, we observed no cytopathic effects (CPEs), and 218 replication levels were very low (no significant increase over control levels on RT-qPCR; data 219 not shown). To further test the effect of KLK13 on replication of HCoV-HKU1 in RD cells, the 220 virus stock was incubated with purified KLK13 (200 nM) and incubated in the presence or 221 absence of a KLK13 inhibitor (10 µM) or DMSO. After 2 h at 32°C pre-treated virus stock was 222 diluted in media as described above and overlaid on RD cells. After 7 days at 32°C we evaluated 223 the presence of the HCoV-HKU1 N sg mRNA. The virus replicated only after treatment with 224 KLK13, and supplementation with the inhibitor blocked this effect infection (Fig. 5C) . 225 author/funder. All rights reserved. No reuse allowed without permission.