Determination of the interactome of non-structural protein12 from highly pathogenic porcine reproductive and respiratory syndrome virus with host cellular proteins using high throughput proteomics and identification of HSP70 as a cellular factor for virus replication.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide. Although extensive research has been focused on PRRSV, the structure and function of some viral proteins like nonstructural protein12 (NSP12), which may play important roles in viral replication and production, still remain unknown. In order to better understand the function of NSP12, we investigated the interaction of NSP12 with cellular proteins using quantitative proteomics coupled with an immune-precipitation strategy based on the over expression of an NSP12-EGFP fusion protein in 293T cells. Data analysis identified 112 cellular proteins interacted with NSP12-EGFP with high probability. The majority of those proteins are nucleic acid binding proteins or chaperones, which are involved in RNA post-transcriptional modification, protein synthesis and cellular assembly and organization. Among them, cellular chaperon HSP70 was verified to interact with PRRSV NSP12 protein, and inhibition of HSP70 significantly reduced the viral mRNA synthesis and virus replication. Our data suggested that NSP12 could recruit cellular proteins such as HSP70 to maintain its own stability and benefit for the virus replication. SIGNIFICANCE: Published data for PRRSV NSP12 is still very limited and the structure and function of NSP12 remain unknown, cellular interactome of PRRSV NSP12 has not been reported to the best of our knowledge. In this paper, we investigated the interaction of NSP12 with cellular proteins using quantitative proteomics coupled with an immune-precipitation strategy, and identified 112 cellular proteins that had a high probability to interact with NSP12. Among these cellular proteins, we verified the interaction of cellular chaperon HSP70 with NSP12, and demonstrated that NSP12 could recruit HSP70 to maintain its own stability and benefit for the virus replication. Our data obtained here could provide crucial clues for better understanding the roles of NSP12 in PRRSV infection.