Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the
fast development of efficient therapeutics. Of the major structural proteins encoded by the SARSCoV-
2 genome, the SPIKE (S) protein has attracted considerable research interest because of the
central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies
aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein
is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous
mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein
have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface
and form a shield-masking specific epitope to escape the virus antigenic recognition. In this
work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana
benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show
that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans
and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana
adopts an overall 3D structure similar to that of recombinant homologues produced in
mammalian cells.