Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43

Abstract

Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with “common colds”, infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells

“…Monoclonal antibody against the SARS-CoV-2 N protein (anti-SARS-CoV-2 N mAb) was prepared from ascitic fluid collected from BALB/c mice injected with hybridoma cells (clone 1G10C4 mAb), as previously described ( Kim et al, 2021a , 2022a , 2022b ). Rabbit anti-SARS-CoV-2 N protein polyclonal antibody (anti-SARS-CoV-2 N Ab, Catalog no.…”

Evaluating the in Vitro Efficacy of Quassinoids from Eurycoma longifolia and Eurycoma harmandiana against Common Cold Human Coronavirus OC43 and SARS-CoV-2 Using In-Cell Enzyme-Linked Immunosorbent Assay

“…The in-cell ELISA method has benefits for high-throughput screening; it requires a shorter analysis time than the conventional antiviral plaque reduction assay, which needs 5–7 days to generate plaques. , In-cell ELISA was used to detect intracellular HCoV-OC43 and SARS-CoV-2 nucleocapsids, thereby assessing the susceptibility of host cells to viral infection and replication processes. The quantitative detection of bulk residues of viral infection in the host cells by the assay implies an inhibitory effect exerted on viral replication by the quassinoid constituents in the roots of EL and EH. − Quassinoid compounds in EL and EH roots exhibited broad anti-coronavirus activity against both HCoV-OC43 and SARS-CoV-2, which seem to have an inhibitory effect on viral replication, similar to that exerted by remdesivir, a novel antiviral drug. In agreement with the literature, javanicin I, a quassinoid from Picrasma javanica bark, inhibited the viral protein R of human immunodeficiency virus-1 (HIV-1) related to HIV replication …”

Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies

“…The nucleotide sequences coding for the SARS-CoV-2 N protein (S clade, nucleotide numbers 28274-29530), MERS-CoV N protein (nucleotide numbers 28566-29804), and HCoV-OC43 N protein (nucleotide numbers 29079-30425) were based on the GenBank IDs MN908947.3, KT029139.1, and NC006213.1, respectively. To obtain recombinant biotin peptide and 6×His-tagged coronavirus N proteins (recombinant SARS-CoV-2 N-Bio-His 6 , MERS-CoV N-Bio-His 6 , and HCoV-OC43 N-Bio-His 6 protein), nucleotide sequences encoding each coronavirus N protein tagged with a biotin peptide and a hexahistidine (6 × His) were synthesized along with restriction enzyme sites (Not I and Kpn I) at the 5′ and 3′ ends, respectively (Bioneer, Daejeon, Republic of Korea), as described previously [ 23 , 29 ]. The synthesized fusion genes were incorporated into a modified pcDNA 3.4 expression vector (Thermo Fisher Scientific) with an IL-2 signal peptide (pcDNA3.4-SARS-CoV-2 N-Bio-His 6 , pcDNA3.4-MERS-CoV N-Bio-His 6 , and pcDNA3.4-HCoV-OC43 N-Bio-His 6 ) for mammalian cell expression in 293F cells.…”

“…To obtain recombinant proteins without biotinylation (recombinant SARS-CoV-2 N-His 6 , MERS-CoV N-His 6 , and HCoV-OC43 N-His 6 protein), the N protein was expressed without the Bir A vector. Each coronavirus N protein was purified from 293F culture supernatants after 5–7 days of cell culture using Ni-NTA-agarose (Qiagen, Hilden, Germany) chromatography as described previously [ 23 , 29 ].…”