Characterization of Two Monoclonal Antibodies That Recognize Linker Region and Carboxyl Terminal Domain of Coronavirus Nucleocapsid Protein.

Xin Zhang,Xin Zhao,Yunnuan Zhu,Jianfei Chen,Hongyan Shi,Da Shi,Hui Dong,Li Feng,Lanying Du

doi:10.1371/journal.pone.0163920

Abstract

The transmissible gastroenteritis virus (TGEV) nucleocapsid (N) protein plays important roles in the replication and translation of viral RNA. The present study provides the first description of two monoclonal antibodies (mAbs) (5E8 and 3D7) directed against the TGEV N protein linker region (LKR) and carboxyl terminal domain (CTD). The mAbs 5E8 and 3D7 reacted with native N protein in western blotting and immunofluorescence assay (IFA). Two linear epitopes, 189SVEQAVLAALKKLG202 and 246VTRFYGARSSSA257, located in the LKR and CTD of TGEV N protein, respectively, were identified after truncating the protein and applying a peptide scanning technique. Using mAb 5E8, we observed that the N protein was expressed in the cytoplasm during TGEV replication and that the protein could be immunoprecipitated from TGEV-infected PK-15 cells. The mAb 5E8 can be applied for different approaches to diagnosis of TGEV infection. In addition, the antibodies represent useful tools for investigating the antigenic properties of the N protein.

Highlights

The four Coronavirus (CoV) genera, alpha, beta, gamma, and deltacoronavirus are clustered in the Coronavirinae subfamily [1, 2]
We described two monoclonal antibodies (mAbs), namely 5E8 against the transmissible gastroenteritis virus (TGEV) N protein linker region (LKR) and 3D7 against the TGEV N protein carboxyl terminal domain (CTD)
The recombinant GST-N protein was expressed in pGEX-TGEV-N-transformed cells

Summary

Introduction

The four Coronavirus (CoV) genera, alpha-, beta-, gamma-, and deltacoronavirus are clustered in the Coronavirinae subfamily [1, 2]. CoVs are pleomorphic, enveloped, single-stranded, positive-sense RNA viruses, with genomes ranging from 26.2 to 31.7 kb [3, 4]. The genomes of CoVs encode four structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). CoV N proteins interact with viral-specific RNAs or RNA intermediates and might play important roles during viral transcription and replication [6, 7]. These proteins might act as RNA chaperones, involved in template switching and required for efficient transcription [8, 9]. Four distinct domains are present in CoV N proteins: intrinsically disordered regions (IDRs), an amino-terminal domain (NTD), a carboxy-terminal

Methods

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Discussion

Conclusion