Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication

Abstract

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication.MethodsThe interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively.ResultsThe interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells.ConclusionOur findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0794-5) contains supplementary material, which is available to authorized users.

Enterovirus 71 Represses Interleukin Enhancer-Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects

“…In this study, we initially show that ILF2 downregulates EV71 TCID50 and attenuates EV71 PFU, thereby repressing EV71 infection. Although this result is similar to previous reports showing that ILF2 regulates the replication of other viruses [28][29][30][31][32][33], this is the first study providing evidence that ILF2 represses EV71 infection. And although ILF2 plays critical roles in the repression of viral infection, the mechanism by which virus antagonizes ILF2-mediated antiviral effects was unknown until this study.…”

“…In addition, 2B interacted with ILF2 ( Figure 4B), and ILF2 associated with 2B ( Figure 4C) in HEK293T cells, confirming the interaction between 2B and ILF2. Since 2B distributes in the cytoplasm [8] while ILF2 mainly locates in the nucleus [33], we next determined whether 2B colocalizes with ILF2 in HEK293T cells. A laser scanning confocal microscope showed that 2B (green) alone was mainly localized in the cytoplasm ( Figure 4D, top panels), and that ILF2 (red) alone was strongly distributed in the nucleus ( Figure 4D, middle panels); however, in the presence of both proteins, 2B and the majority of ILF2 were colocalized in the cytoplasm to form spots ( Figure 4D, bottom panels).…”

“…ILF2 also plays a role in the progression of pancreatic carcinoma [21,22], hepatocellular carcinoma [20,23], lung cancer [24], esophageal squamous cell carcinoma [25], gastric cancer [26], and breast cancer [27]. Moreover, ILF2 regulates the replication of immunodeficiency virus type 1 [28], hepatitis C virus [29], rhinovirus type 2 [30], white spot syndrome virus [31], human papilloma virus [32], and respiratory syndrome virus [33]. However, the mechanisms underlying the control of ILF2-mediated antiviral effects have not been reported, and the role of ILF2 in EV71 replication is unknown.…”

Nucleotide-binding oligomerization domain-like receptor X1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral Nsp9

“…On the one hand, interaction between Nsp9 and cellular annexin A2, retinoblastoma protein (pRb), DDX5 positively regulates the replication of PRRSV in vitro, demonstrating that PRRSV heavily relies on host cellular proteins to complete life cycle (Dong et al, 2014;Li et al, 2014a;Zhao et al, 2015). On the other hand, recent literature indicated that the interaction of Nsp9 with SUMO E2 conjugating enzyme Ubc9 and cellular protein interleukin-2 enhancer binding factor 2 (ILF2) through its RdRp domain resulted in a significantly decrease of virus titers, indicating that cells utilize host antiviral factors as defense mechanisms to limit PRRSV infection Wen et al, 2017). Altogether, these results provide insights into the complex interactions of PRRSV RdRp with the host proteome.…”

Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

“…PRRSV nsp-specific antibodies. The mouse MAbs specific for PRRSV nsp1␤, nsp2 (E3G11), nsp4, nsp9, nsp10 (4D9), and nsp11 (3F9) were prepared in our laboratory and have been reported previously (72)(73)(74)(75)(76). Mouse anti-nsp12 MAb (1E5) was kindly provided by Changjiang Weng (Harbin Veterinary Research Institute) (57).…”