Abstract
BackgroundThe highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) continues to pose one of the greatest threats to the swine industry. M protein is the most conserved and important structural protein of PRRSV. However, information about the host cellular proteins that interact with M protein remains limited.MethodsHost cellular proteins that interact with the M protein of HP-PRRSV were immunoprecipitated from MARC-145 cells infected with PRRSV HuN4-F112 using the M monoclonal antibody (mAb). The differentially expressed proteins were identified by LC-MS/MS. The screened proteins were used for bioinformatics analysis including Gene Ontology, the interaction network, and the enriched KEGG pathways. Some interested cellular proteins were validated to interact with M protein by CO-IP.ResultsThe PRRSV HuN4-F112 infection group had 10 bands compared with the control group. The bands included 219 non-redundant cellular proteins that interact with M protein, which were identified by LC-MS/MS with high confidence. The gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway bioinformatic analyses indicated that the identified proteins could be assigned to several different subcellular locations and functional classes. Functional analysis of the interactome profile highlighted cellular pathways associated with protein translation, infectious disease, and signal transduction. Two interested cellular proteins—nuclear factor of activated T cells 45 kDa (NF45) and proliferating cell nuclear antigen (PCNA)—that could interact with M protein were validated by Co-IP and confocal analyses.ConclusionsThe interactome data between PRRSV M protein and cellular proteins were identified and contribute to the understanding of the roles of M protein in the replication and pathogenesis of PRRSV. The interactome of M protein will aid studies of virus/host interactions and provide means to decrease the threat of PRRSV to the swine industry in the future.
Highlights
The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-Porcine reproductive and respiratory syndrome virus (PRRSV)) continues to pose one of the greatest threats to the swine industry
The expression of M protein upon PRRSV infection MARC-145 cells were infected with the HuN4-F112 and collected at 12 h to 84 h post-infection to detect the expression of M protein arising from PRRSV infection
Identification of host cellular proteins that interact with PRRSV M protein MARC-145 cells were infected with HuN4-F112/HuN4F5 at an Multiplicity of infection (MOI) of 0.1 to efficiently precipitate M protein and subsequently identify host proteins that interact with M protein
Summary
The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) continues to pose one of the greatest threats to the swine industry. The rest of the ORFs of PRRSV encode eight structural proteins: GP2, E, GP3, GP4, GP5, M, N, and ORF5a [20, 25, 26]. The M/GP5 complex was identified as a ligand for sialoadhesin, which is involved in the entry process of PRRSV in to PAMs [35, 36]. These findings reveal that the M protein is involved in PRRSV infection and immunity and the entry process of the pathogen. The molecular mechanisms of its involvement in these functions have not been elucidated clearly
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