Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus

Abstract

Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.