Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage Display Technology.

Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues 82GELPPNDTPATTRVT96 of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.

Structural analysis, virtual screening and molecular simulation to identify potential inhibitors targeting 2'-O-ribose methyltransferase of SARS-CoV-2 coronavirus.

SARS-CoV-2, an emerging coronavirus, has spread rapidly around the world, resulting in over ten million cases and more than half a million deaths as of July 1, 2020. Effective treatments and vaccines for SARS-CoV-2 infection do not currently exist. Previous studies demonstrated that nonstructural protein 16 (nsp16) of coronavirus is an S-adenosyl methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase) that has an important role in viral replication and prevents recognition by the host innate immune system. In the present study, we employed structural analysis, virtual screening, and molecular simulation approaches to identify clinically investigated and approved drugs which can act as promising inhibitors against nsp16 2'-O-MTase of SARS-CoV-2. Comparative analysis of primary amino acid sequences and crystal structures of seven human CoVs defined the key residues for nsp16 2-O'-MTase functions. Virtual screening and docking analysis ranked the potential inhibitors of nsp16 from more than 4,500 clinically investigated and approved drugs. Furthermore, molecular dynamics simulations were carried out on eight top candidates, including Hesperidin, Rimegepant, Gs-9667, and Sonedenoson, to calculate various structural parameters and understand the dynamic behavior of the drug-protein complexes. Our studies provided the foundation to further test and repurpose these candidate drugs experimentally and/or clinically for COVID-19 treatment.Communicated by Ramaswamy H. Sarma.

Repurposing Therapeutics to Identify Novel Inhibitors Targeting 2'-O-Ribose Methyltransferase Nsp16 of SARS-CoV-2 (preprint)

Three coronaviruses (CoVs): severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently identified SARS-CoV-2 in December 2019, have caused deadly pneumonia in humans since the beginning of the 21st century. The SARS-CoV-2 causes coronavirus disease-19 (COVID-19) with influenza-like symptoms ranging from mild discomfort to severe lung injury and multi-organ failure, eventually leading to death. As of April 30, 2020, more than three million (3,175,207) COVID-19 cases were reported worldwide, and more than 220,000 (224,172) patients have died (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). Effective treatments and vaccines for SARS-CoV-2 infection do not currently exist. Thus, it will be of great benefit to identify and repurpose already well-characterized compounds and approved drugs for use in combating COVID-19. CoVs are positive-sense RNA viruses that replicate in the cytoplasm of infected cells. Replication and transcription of the CoV RNA genome are achieved by a complex RNA replication/transcription machinery, consisting of at least 16 viral nonstructural proteins (nsp). Previous studies demonstrated that nsp16 proteins of SARS-CoV-1 and MERS-CoV have methyltransferase (MTase) activities that catalyze methylation of the first transcribed nucleotide at the ribose 2’-O position (2’-O-Me). The 2’-O-Me of virus cap RNAs protects itself from degradation by 5′-3′ exoribonucleases, ensures efficient translation, and helps to prevent recognition by the host innate immune system. The importance of nsp16 2'-O-MTase activity for CoV infection and pathogenesis was previously documented by in vitro and in vivo studies. For SARS-CoV-1, the absence of nsp16 2′-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. In addition, nsp16 down-regulates the activities of innate immune sensing factors: retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 protein (MDA5). Thus, inhibition of nsp16 2’-O-MTase activities should restrain viral replication and enable recognition by the host innate immune system, making the nsp16-MTase a promising target for the identification of new anti-SARS-CoV-2 drugs. In the present study, we employed structural analysis, virtual screening, and systematic drug repurposing approaches to identify “approved” drugs which can act as promising inhibitors against nsp16 2′-O-MTase of SARS-CoV-2. We first performed comparative analysis of primary amino acid sequences and crystal structures of seven human CoVs and defined the key residues for nsp16 2-O’-MTase functions. From the virtual screening against nsp16 2′-O-MTase of SARS-CoV-2, we provide a ranking of the predicted binding affinities of 1,380 top hit compounds corresponding to 967 “approved” drugs. Furthermore, we have calculated various structural parameters of our top-ranking drugs. Our studies provided the foundation to further test and repurpose these candidate drugs experimentally and clinically for COVID-19 treatment.