White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag

Abstract

ABSTRACT Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twenty-eight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, ≥20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.