Interactome Profile of the Host Cellular Proteins and the Nonstructural Protein 2 of Porcine Reproductive and Respiratory Syndrome Virus

Li Wang,Xin Guo,Xinna Ge,Hanchun Yang,Lei Zhou,Yan Li,Kangzhen Yu,Han Zhang

doi:10.1371/journal.pone.0099176

Abstract

The nonstructural protein 2 (NSP2) is considered to be one of crucial viral proteins in the replication and pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV). In the present study, the host cellular proteins that interact with the NSP2 of PRRSV were immunoprecipitated with anti-Myc antibody from the MARC-145 cells infected by a recombinant PRRSV with 3xMyc tag insertion in its NSP2-coding region, and then 285 cellular proteins interacting with NSP2 were identified by LC-MS/MS. The Gene Ontology and enriched KEGG Pathway bioinformatics analyses indicated that the identified proteins could be assigned to different subcellular locations and functional classes. Functional analysis of the interactome profile highlighted cellular pathways associated with infectious disease, translation, immune system, nervous system and signal transduction. Two interested cellular proteins–BCL2-associated athanogene 6 (BAG6) and apoptosis-inducing factor 1 (AIF1) which may involve in transporting of NSP2 to Endoplasmic reticulum (ER) or PRRSV-driven apoptosis were validated by Western blot. The interactome data between PRRSV NSP2 and cellular proteins contribute to the understanding of the roles of NSP2 in the replication and pathogenesis of PRRSV, and also provide novel cellular target proteins for elucidating the associated molecular mechanisms of the interaction of host cellular proteins with viral proteins in regulating the viral replication.

Highlights

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease, causing great economic losses to the swine industry worldwide [1,2]
In order to label the nonstructural protein 2 (NSP2) and facilitate to identify the host cellular proteins interacted with NSP2, the region–aa 338 to aa 367 of Porcine reproductive and respiratory syndrome virus (PRRSV) JXwn06 NSP2 was replaced with 3xMyc tag in the infectious clone plasmid pWSK-JXwn
To confirm the successful recovery of virus and examine the stability of the foreign tag in the virus, the chimeric virus designated as RvMyc-JXwn and its parental virus RvJXwn were serially passaged in MARC-145 cells, and the confocal microscopy analysis (Figure 1A) with Myc polyclonal antibody and PRRSV N protein monoclonal antibody and the sequence analysis of the NSP2-coding region including Myc tag gene were performed

Summary

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease, causing great economic losses to the swine industry worldwide [1,2]. This disease was first described as ‘‘mystery swine disease’’ in the United states in 1987 [3], which is characterized by severe reproductive failure in sows and respiratory disorders in all age of pigs [3,4]. The viral genome is approximately 15 kb in length and encodes at least ten open reading frames (ORFs) comprising of ORF1a, ORF1b, ORF2a, ORF2b, ORFs3-7 and the recently discovered ORF5a [8,9]. ORF1a and ORF1b comprised approximately two-third of the genome encode the replicase polyproteins which are considered to be autoproteolytically cleaved into at least 14 non-structural proteins (NSPs) including the newly discovered transframe fusion (TF) in NSP2-coding region [10,11]

Methods

Results

Discussion

Conclusion