Structural Characterization of Human Coronavirus NL63 N Protein.

Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of virus structure assembly. The results expand our understanding of coronavirus physiology and may facilitate future efforts to control the associated infections.

Crystal structures of human coronavirus NL63 main protease at different pH values.

Human coronavirus NL63 (HCoV-NL63), which belongs to the genus Alphacoronavirus, mainly infects children and the immunocompromized and is responsible for a series of clinical manifestations, including cough, fever, rhinorrhoea, bronchiolitis and croup. HCoV-NL63, which was first isolated from a seven-month-old child in 2004, has led to infections worldwide and accounts for 10% of all respiratory illnesses caused by etiological agents. However, effective antivirals against HCoV-NL63 infection are currently unavailable. The HCoV-NL63 main protease (Mpro), also called 3C-like protease (3CLpro), plays a vital role in mediating viral replication and transcription by catalyzing the cleavage of replicase polyproteins (pp1a and pp1ab) into functional subunits. Moreover, Mpro is highly conserved among all coronaviruses, thus making it a prominent drug target for antiviral therapy. Here, four crystal structures of HCoV-NL63 Mpro in the apo form at different pH values are reported at resolutions of up to 1.78 Å. Comparison with Mpro from other human betacoronaviruses such as SARS-CoV-2 and SARS-CoV reveals common and distinct structural features in different genera and extends knowledge of the diversity, function and evolution of coronaviruses.

The SARS-CoV-2 Exerts a Distinctive Strategy for Interacting with the ACE2 Human Receptor.

The COVID-19 disease has plagued over 200 countries with over three million cases and has resulted in over 200,000 deaths within 3 months. To gain insight into the high infection rate of the SARS-CoV-2 virus, we compare the interaction between the human ACE2 receptor and the SARS-CoV-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. SARS-CoV, SARS-CoV-2, and HCoV-NL63 recognize ACE2 as the natural receptor but present a distinct binding interface to ACE2 and a different network of residue-residue contacts. SARS-CoV and SARS-CoV-2 have comparable binding affinities achieved by balancing energetics and dynamics. The SARS-CoV-2-ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to the SARS-CoV-ACE2 complex. These findings expose an exceptional evolutionary exploration exerted by coronaviruses toward host recognition. We postulate that the versatility of cell receptor binding strategies has immediate implications for therapeutic strategies.

Automatically Fixing Errors in Glycoprotein Structures with Rosetta.

Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins that is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup, and refinement of glycan-containing structures. We benchmarked this approach using 12 crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.

Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy.

The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.

Crystallization and preliminary crystallographic study of human coronavirus NL63 main protease in complex with an inhibitor.

Human coronavirus NL63 mainly infects younger children and causes cough, fever, rhinorrhoea, bronchiolitis and croup. It encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of human coronavirus NL63 was crystallized in complex with a Michael acceptor. The complex crystals diffracted to 2.85 Šresolution and belonged to space group P41212, with unit-cell parameters a = b = 87.2, c = 212.1 Å. Two molecules were identified per asymmetric unit.

[Prokaryotic expression and characterization of two recombinant receptor-binding domain(RBD) proteins of human coronavirus NL63(HcoV-NL63)].

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.

[Characterization and development of recombinant vaccinia viruses expressing different segments of spike protein derived from human coronavirus NL-63].

The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.