The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed. Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

To date, seven identified coronaviruses (CoVs) have been found to infect humans; of these, three highly pathogenic variants have emerged in the 21st century. The newest member of this group, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected at the end of 2019 in Hubei province, China.

The Coronaviridae family clusters a number of large RNA viruses, which share several structural and functional features. However, members of this family recognize different cellular receptors and exploit different entry routes, what affects their species specificity and virulence. The aim of this study was to determine how human coronavirus OC43 enters the susceptible cell. Using confocal microscopy and molecular biology tools we visualized early events during infection. We found that the virus employs caveolin-1 dependent endocytosis for the entry and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Furthermore, the vesicle internalization process requires actin cytoskeleton rearrangements. With our research we strove to broaden the understanding of the infection process, which in future may be beneficial for the development of a potential therapeutics.

Despite high similarity of canine respiratory coronavirus (CRCoV), bovine coronavirus, (BCoV) and human coronavirus OC43 (HCoV-OC43), these viruses differ in species specificity. For years it was believed that they share receptor specificity, utilizing sialic acids for cell surface attachment, internalization, and entry. Interestingly, careful literature analysis shows that viruses indeed bind to the cell surface via sialic acids, but there is no solid data that these moieties mediate virus entry. In our study, using a number of techniques, we showed that all three viruses are indeed able to bind to sialic acids to a different extent, but these molecules render the cells permissive only for the clinical strain of HCoV-OC43, while for others they serve only as attachment receptors. CRCoV and BCoV appear to employ human leukocyte antigen class I (HLA-1) as the entry receptor. Furthermore, we identified heparan sulfate as an alternative attachment factor, but this may be related to the cell culture adaptation, as in ex vivo conditions, it does not seem to play a significant role. Summarizing, we delineated early events during CRCoV, BCoV, and HCoV-OC43 entry and systematically studied the attachment and entry receptor utilized by these viruses.

Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation. IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.