The characterisation of human coronavirus nl63 proteins

Abstract

Human Coronavirus NL63 (HCoV-NL63) is one of seven coronaviruses (CoVs) that cause respiratory disease in the global population. The Membrane (M) and Nucleocapsid (N) proteins are part of the core CoV-structural proteins, crucial in viral replication and virion assembly. Here the expression of HCoVNL63 M and N was characterized across multiple in vitro systems including bacterial, insect and mammalian. To detect untagged proteins in viral structural studies, anti-peptide antibodies were generated in a mouse model. Polyclonal antisera and hybridoma-secreted antibodies exhibited specific binding to their respective full length protein antigens. Anti-peptide monoclonal antibodies were successfully generated against the HCoV-NL63 M and N proteins. During CoV infection, the interaction of CoV M and N is necessary for the production of infectious virions. For the first time, co-expressed, full length HCoV-NL63 M and N were assayed for protein-protein interaction in a mammalian cell system, allowing for native protein folding and modification. M protein formed higher order homomultimers in the presence and absence of co-expressed N. Complexed M and N were co-purified from mammalian cells and confirmed as interaction partners, even under denaturing conditions. HCoVNL63 M and N proteins formed stable interactions when co-expressed in vitro. The strong M-N association in the absence of other viral components highlights the importance of this interaction during virus replication in the host cell. The subcellular localization of HCoV-NL63 N was evaluated from early to late expression, and N exhibited no nucleocytoplasmic shuttling during protein synthesis and maturation. The identified pat4/pat7 nuclear localisation motifs within N may have been inactive; alternatively the localization of N expressed alone was modulated by multiple directive signals provided within the protein. A recombinant Baculovirus was used to express HCoV-NL63 M, towards examining CoV structural protein dynamics in an alternate eukaryotic insect system. Preliminary results indicated that M was expressed at 48-72 hours post-infection. CoV M and N each play multifunctional roles in the viral life cycle, and are implicated in the host-immune response. Investigating the relationships between these and other CoV structural components brings us closer to understanding CoV replication. Important viral protein-protein interactions could present new targets for exploitation in the hunt for an effective anti-coronaviral agent. HCoV-NL63 has been a human pathogen for over 500 years, and continues to adapt genetically, retaining viral fitness in the environment. Able to cause both upper and lower respiratory tract infections, HCoV-NL63 remains a clinically relevant and interesting HCoV species, that may hold the secrets to viral persistence and seasonal infection patterns of endemic HCoVs.