Collected fractions were analyzed by dot blotting with anti-M antibody. (C) Purification profiles of MEN and MENS VLPs on a heparin column. M, N, and S are lanes from blots detected with anti-M, anti-N, and anti-S antibodies, respectively. Culture medium was harvested from insect cells infected with (M+E) rBV, N rBV, and S rBV. (B) As anti-S antibody could not be used for immunostaining, S protein incorporation was demonstrated by Western blotting. N protein was detected with mouse monoclonal antibody and is immunostained in green, whereas M protein was detected with rabbit polyclonal serum and is immunostained in red. (A) Fluorescence microscope images of insect cells expressing N and M proteins. 1D).įIG 1 FIG 1 Production of HCoV-NL63 VLPs. The analysis of purified samples by transmission electron microscopy (TEM) showed spherical particles of different diameters, which might reflect the amount of the N protein incorporated into VLPs ( Fig. Both VLPs, composed of M, E, and N (MEN) and M, E, N, and S (MENS) proteins, were concentrated and purified using a heparin column according to a previously developed protocol ( 22) ( Fig. In contrast, the apparent molecular weight of the S protein band was higher than expected, most likely because of the glycosylation and/or incomplete denaturation of the protein trimer. Similarly, two bands of the N protein were observed, upon electrophoresis, resulting from protein degradation under denaturing conditions ( 23). In agreement with our previous studies, the M protein migrated as two bands, reflecting its two different glycosylation states. The M (26-kDa), N (42-kDa), and S (150-kDa) proteins were detected in the secreted fraction when insect cells were coinfected with (M+E) rBV, N rBV, and, optionally, S rBV ( Fig. To verify whether VLPs were effectively assembled and released, the culture medium harvested from insect cells expressing M, E, N, and S proteins was analyzed by Western blotting. Colocalization of the M and N proteins was observed, suggesting the formation of a protein complex within the producer cells ( Fig. To evaluate protein expression, insect cells infected with bicistronic recombinant baculovirus (rBV) coding for M and E proteins (M+E) and monocistronic N rBV (and optionally monocistronic S rBV) were immunostained and examined under a confocal microscope. HCoV-NL63 VLPs composed of the M, E, N, and (optionally) S proteins were produced in insect cells, as previously described ( 22), with the modification that the N protein was also included. This is illustrated by HSPG binding of the Tat protein of human immunodeficiency virus, which after internalization activates transcription of the viral RNA ( 21). HSPGs can also enhance virulence by binding accessory viral factors necessary for viral replication. Furthermore, the majority of oncogenic viruses (hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papillomaviruses, Merkel cell polyomavirus, and human T cell lymphotropic virus type 1) initially attach to the HSPGs ( 20). As an example, binding to HSPG induces structural rearrangements of proteins responsible for infection by adeno-associated virus 2 ( 16), adenovirus types 2 and 5 ( 17), human papillomavirus 16 ( 18), and several herpesviruses ( 19). Binding to HSPG is the initial event promoting subsequent recognition of a secondary receptor by increasing the local concentration of pathogens or triggering conformational changes of proteins involved in viral entry. HSPGs are glycoproteins that are ubiquitous at the surface of the mammalian cell. Furthermore, several alpha-, beta-, and gammacoronaviruses, e.g., TGEV, human coronavirus HKU1, HCoV-OC43, bovine respiratory coronavirus (BCoV), and avian infectious bronchitis virus (IBV), reportedly use sialic acids for initial attachment to the cell (summarized in reference 10).
A number of receptors have been described for betacoronaviruses, including carcinoembryonic antigen-related cell adhesion molecule 1 for murine hepatitis virus (MHV), dipeptidyl peptidase 4 for Middle East respiratory syndrome coronavirus (MERS-CoV), and major histocompatibility complex class I for human coronavirus OC43 ( 5 – 9). At the same time, ACE2 serves as a receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), a betacoronavirus ( 4). Some alphacoronaviruses, e.g., transmissible gastroenteritis coronavirus (TGEV) and feline infectious peritonitis coronavirus (FIPV), employ aminopeptidase N, whereas human coronavirus NL63 (HCoV-NL63) utilizes angiotensin-converting enzyme 2 (ACE2) ( 1 – 3).
The tissue and species specificities are determined by the presence of appropriate adhesion and entry receptors on the cell surface. Coronaviruses (CoVs), enveloped RNA viruses, infect a wide variety of species, causing various clinical conditions.