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Caractérisation de la protéine S du coronavirus humain 229E

Abstract : The human coronavirus 229E (HCoV-229E) is a causative agent of common colds and can lead to severe respiratory complications in elderly persons and those with underlying disease. Coronavirus are enveloped viruses with a single stranded, positive-sense RNA genome. Three viral proteins are anchored in the viral enveloppe : the spike (S) protein, the membrane (M) protein and the enveloppe (E) protein. The M and E proteins are involved in viral assembly and secretion. The spike proteins assemble into trimers at the surface of the virions and play a key role in the early steps of viral infection. The spike protein comprised two domains, the S1 domain responsible for receptor binding and the S2 domain responsible for fusion of the viral enveloppe with the host cell membrane. Coronavirus fusion is activated by the proteolytic processing of the spike protein. First, we charaterized the proteolytic processing of the HCoV-229E spike protein by trypsin-like serine-proteases. To do so, we first cloned the spike protein of a circulating isolate of HCoV-229E. To investigate the role of the S1/S2 junction and the specific role of the 3 arginine residues located in the S2’ region in the proteolytic activation of HCoV-229E spike protein, the arginine residues present at these positions were mutated individually (R565N, R679N, R683N or R687N). Our results show that unlike other coronaviruses, HCoV-229E fusion activation appears to be a one step process. Indeed, the cleavage of the S1/S2 interface does not seem to be a pre-requisite, and the fusion activation strongly relies on the S2’ region, with R683 acting as the cleavage site.The spike protein is highly N-glycosylated and is the main target of neutralizing antibodies. We analysed the role of S1 domain N-glycosylation in the entry functions of the S protein and in neutralization by antibodies. Analysis of the sequence of the cloned protein shows the presence of 33 potential N-glycosylation sites, 18 being located in the S1 domain (numbered from N1 to N18). We mutated the 18 N-glycosylation sites of S1 individually by site-directed mutagenesis and studied the effect of the mutations using retroviral pseudotyped particles. Infectivity of the spike proteins with mutation either at the N6, N7 or N9 glycosylation site was strongly impaired. We did not detect any difference of interaction of these mutants with the soluble form of the receptor, the aminopeptidase N (APN). Results obtained by inducing the fusion of pseudoparticles at the cell surface with trypsin suggest that N-glycans located at the position N6, N7 and N9 are involved in viral fusion. However, the proteolytic processing of the protein required for fusion activation does not seem to be affected. Two mutants N12 and N15 show an increase of infectivity. Mutation of the N-glycosylation site N17 induces a partial decrease in infectivity. Indeed a decrease of spike protein incorporation into pseudoparticles was observed likely due to misfolding of the protein as shown by the profile of the mutant in western blot under reducing and non-reducing conditions. We next assessed if N-glycans can modulate the recognition of the spike protein by neutralizing antibodies. Pseudoparticles harbouring the different mutants were produced and used to infect cells in presence or absence of neutralizing antibodies. Our data demonstrate that mutants N4, N10, N11, N12, N15, N16, N17, N18 reduce the sensitivity of pseudoparticules to antibody neutralization. Taken together our results suggest that N-glycans of the S protein play an important role in viral entry and modulate the recognition of the protein by neutralizing antibodies.
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Submitted on : Monday, September 2, 2019 - 1:08:20 AM
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  • HAL Id : tel-02275786, version 1



Ariane Bonnin. Caractérisation de la protéine S du coronavirus humain 229E. Médecine humaine et pathologie. Université de Lille, 2018. Français. ⟨NNT : 2018LILUS007⟩. ⟨tel-02275786⟩



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