Interactome profile of the host cellular proteins and the nonstructural protein 2 of porcine reproductive and respiratory syndrome virus

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.

Figure 1. Identification of the rescued recombinant PRRSV with the 3xMyc-tag.

(A) The confocal microscopy analysis of fifth passage of the recombinant virus RvMyc-JXwn and its parental virus RvJXwn of the passage 5 and 10 with anti-PRRSV N protein monoclonal antibodies (SDOW17) and anti-Myc polyclonal antibody. (B) In vitro growth kinetics of the fifth passage of the recombinant virus RvMyc-JXwn and its parental virus RvJXwn were drawn by assaying the viral titers of the supernatants harvested from 12 h to 120 h post-infection using microtitration infectivity assays. Data are means±standard deviations (error bars) from three independent trials. No significant difference between the recombinant virus and the parental virus (p>0.05).

Figure 2. The expression of PRRSV NSP2.

Cell lysates from RvMyc-JXwn-infected MARC-145 cells at different time points were subjected to Western blot with anti-Myc antibody and anti-β-actin antibody.

Figure 3. Identification of the cellular proteins that interact with PRRSV NSP2 by immunoprecipitation (IP).

Cell lysates from RvMyc-JXwn- or RvJXwn-infected MARC-145 cells were immunopreciptiated with anti-Myc antibody, and subsequently the immunoprecipitated proteins were separated both by 8% (A) and 15% (B) SDS-PAGE and visualized by sliver staining. Asterisks indicate the protein bands of IgG heavy chain with 55 KDa or IgG light chain with 26 KDa. The numbers on the right lane show the differential protein bands between RvMyc-JXwn- and RvJXwn-infected MARC-145 cells.

Figure 4. The annotation of proteins interacting with PRRSV NSP2 using Gene Ontology.

(A) Biological process. (B) Cellular components. (C) Molecular function.

Figure 5. Classification of the enriched KEGG Pathways of the cellular proteins interacting with PRRSV NSP2.

Figure 6. The interaction network of the identified proteins with BAG6 and AIF1.

Figure 7. Confirmation of the interaction of PRRSV NSP2 with BAG6 and AIF1.

(A to D) The interaction of NSP2 and exogenous BAG6 (A, C) and AIF1 (B, D). 293FT cells were co-transfected with 6 µg of the indicated plasmids. Cell lysates were prepared at 24 h after transfection and the proteins were immunoprecipitated with anti-HA or anti-FLAG antibodies. Proteins in cell lysates (input) and immunoprecipitated samples were detected with the antibodies against FLAG and HA by Western blot. (E) The interaction of NSP2 with endogenous BAG6 and AIF1. MARC-145 cells were infected with the recombinant virus RvMyc-JXwn and its parental virus RvJXwn. Cell lysates were prepared at 48 h post-infected and subjected to IP with anti-Myc antibody, the immunoprecipitated samples were detected with the antibodies against BAG6 and AIF1 by Western blot.