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.

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.

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.