Molecular Mechanisms Of The Interaction Of PRRSV Nsp2with Host Cellular Proteins BAG6and AIF1

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important etiological agents affecting swine industry worldwide. The nonstructural protein2(Nsp2) of PRRSV is the largest viral protein that plays important roles in the PRRSV infection. In the present study, the host cellular proteins that interact with the Nsp2of PRRSV were first identified in the MARC-145cells and an interactome profile between them was drawn; Next, the molecular mechanisms of the interaction of Nsp2with the host cellular proteins, BCL2-associated athanogene6(BAG6) and apoptosis-inducing factor1(AIF1), and the roles of their interactions in the PRRSV replication were explored. The objective of this study is to provide scientific evidences for elucidating the biological fuctions of Nsp2during the PRRSV infection.The infectious cDNA clone of JXwn06was used to generate a chimeric cDNA clone that contains3×Myc tag insertion in its Nsp2-coding region. A recombinant virus was rescued from the chimeric cDNA clone and named RvMyc-JXwn. The host cellular proteins that interact with the Nsp2of PRRSV were immunoprecipitated from the MARC-145cells infected with RvMyc-JXwn with an anti-Myc antibody, and617cellular proteins interacting with Nsp2were then identified by LC-MS/MS. Out of these proteins,285proteins with high Confidence Icons (P<0.01) were used for bioinformatics analysis. Functional analysis of the interactome profile indicated that these proteins were involved in the cellular pathways associated with infectious disease, translation, immune system, nervous system and signal transduction.Given to the fuctions of Nsp2and cellular proteins, we explored the molecular mechanisms of the interaction between Nsp2and cellular proteins-BAG6and AIF1. The binding regions of BAG6and Nsp2were identified by Co-IP. Our results showed that the N-terminus but not the cysteine protease (CP) active sites of Nsp2interacted with the N-terminal ubiquitin-like (UBL) domain of BAG6. The confocal microscopy analysis showed that BAG6localized in the nucleus of the cells re-localized to the cytoplasm and co-localized with Nsp2following PRRSV infection in MARC-145cells or PAMs or Nsp2-expressing plasmid transfection in MARC-145cells. Subsequently, we demonstrated that BAG6contributed to target Nsp2to the endoplasmic reticulum (ER)-originated double-membrane vesicles (DMVs). Additionally, the ER stress-mediated apoptosis was discovered in PRRSV-infected cells, and Nsp2induced the ER stress-mediated apoptosis by deubiquitinating BAG6-bound defective proteins and preventing them from degrading, which resulted in the accumulation of defective protein during PRRSV infection. Finally, we found that BAG6played a crucial role in promoting the PRRSV replication, whereas it did not affect the virus release.The binding regions of AIF1and Nsp2were identified by Co-IP. The results showed that the N-terminus of Nsp2interacted with AIF1, whereas the CP active sites were necessary for this interaction. The confocal microscopy analysis indicated that Nsp2could co-localize with AIF1in the cytoplasm and lead to the nuclear translocation of AIF1following PRRSV infection in MARC-145cells or PAMs or Nsp2-expressing plasmid transfection in MARC-145cells. We also found that Nsp2could stabilize AIF1by preventing proteasome from degrading it. In the present study, we demonstrated for the first time that PRRSV infection and Nsp2expression could induce the caspase-independent apoptosis mediated by AIF1. In addition, our data suggested that AIF1played a key role in generating ATP during PRRSV infection and enhanced the replication of PRRSV.As a whole, we identified the host cellular proteins that interact with the Nsp2of PRRSV and drawn an interactome profile of Nsp2with host cellular proteins, providing important clues and evidence for exploring the interaction between Nsp2and cellular proteins. We revealed the molecular mechanisms affecting viral replication by the interaction of PRRSV Nsp2with BAG6or AIF, providing an insight and novel way for further exploration of the biological functions and molecular mechanisms of Nsp2which is involved in the replication and pathogenesis of PRRSV.