Interactions Of PRRSV Nsp2,Nsp3,E With Host Cellular Proteins CD2AP, IFITM1, Tetherin And Their Biological Significances

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens affecting swine industry in China. It is of significance to explore the interactions among viral proteins and host cellular proteins, which helping to understand the pathogenesis of PRRSV. In the present study, the interactions among viral protein and host cellular proteins were screened, and the molecular mechanisms and biological significances of the interaction between Nsp3and IFITM1, E and Tetherin, Nsp2and CD2AP were further analyzed, in order to provide scientific evidences for elucidating the pathogenesis of PRRSV.The recombinant plasmids pGBKT7with respective genes of PRRSV (except for Nsp6and Nsp8) were transformed into Y2H Gold to examine autoactivation and toxicity of each viral protein. The results showed that the proteins of PRRSV had no effect for autoactivation and toxicity. Potential cellular proteins interacting with Nsp2, Nsp9, Nsp10, Nsp11, and Nsp12of PRRSV were screened by a cDNA library derived from pulmonary alveolar macrophages (PAMs) and the yeast two-hybrid system. The final possible targeting proteins were identified by extraction of plasmid from the positive clones, sequence analysis and sequence alignment in NCBI. In addition, cDNA fragments of intrinsic virus-restriction factors including IFITM1, IFITM3, Tetherin, ISG15, ISG56, Mxl, PKR and OAS were amplified from PAMs, and then cloned into pGADT7as prey protein. To analyze the interaction between PRRSV proteins and intrinsic restriction factors, respective bait (pGBKT7) and prey (pGADT7) plasmids were co-transformed into the yeast Y2H Gold cells. The results indicated the interaction between IFITM1and Nsp3, Tetherin and E, respectively.The interactions of Nsp3and IFITM1, E and Tetherin were confrimed by Co-IP. Confocal immunofluorescence analyses revealed that, in PRRSV-infected MARC-145cells and the transfected cells with the IFITM1-or Tetherin-expressing plasmid, IFITM1was shown to be mainly distributed perinuclear, and Tetherin was proposed to be partially removed away from cell surface. Moreover, the overexpression of IFITM1or Tetherin was shown to have no obvious effects on the replication of PRRSV in MARC-145cells. The Nsp3of PRRSV was further demonstrated to induce the proteasome-dependent degradation of IFITM1upon PRRSV infection. These findings suggest that PRRSV might counteract the antiviral functions of IFITM1and Tetherin by the interaction of the Nsp3with IFITM1and the E protein with Tetherin, providing a novel clue for exploring possible mechanisms associated with the evasion of PRRSV from immune recognition of host.The interaction of Nsp2with exogenous or endogenous CD2AP was demonstrated by Co-IP, and the binding of the proline-rich residues of Nsp2to the SH3domains of CD2AP was further determined by yeast two-hybrid system. The Nsp2was shown to co-localize with CD2AP in the perinuclear pattern by confocal immunofluorescence. Analyses of protein expression in PRRSV-infected MARC-145cells showed that Nsp2could induce the degradation of CD2AP by up-regulating the CatL expression, and the siRNA-mediated silencing of CD2AP gene in MARC-145cells resulted in a significant reduction of the virus titer and the expression of Nsp2in cells. The effect of CD2AP on cytoskeletion and apoptosis was further explored. The results showed that Nsp2induced the cytoskeleton and degradation of CD2AP leading to the disruption of normal actin the influence of cell division, and the siRNA specific for CD2AP was beneficial to the production of TGF-β1in the early stages of PRRSV infection. In addition, the Nsp2could up-regulate TGF-β1-induced apoptosis by activating TGF-β1/Smad3signaling pathway. In this process, cellular dendrins were transfered to the nucleus, which promoted expression of cytosolic CatL and TGF-β1-induced apoptosis. Finally, immunohistochemical examinations showed that the expressions of cytoskeleton and apoptosis were related to effective moleculars TGF-β1and CatL, which primarily located in alveolar and bronchial epithelial cell and pulmonary macrophages in the lungs of PRRSV-infected pigs. The above results revealed that Nsp2contributes to cellular pathogenesis of PRRSV, providing a scientific basis for elucidating the partly cellular pathological changes caused by PRRSV infection.In summary, our study revealed the biological significances of the interaction between Nsp2and CD2AP, Nsp3and IFITM1, E and Tetherin, respectively. Our findings provide valuable and essential basis for understanding and elucidating the molecular pathogenesis of PRRSV.