| Promyelocytic leukaemia nuclear bodies (PML-NBs) are dynamic intranuclear structures in mam-malian cells. PML is the organizer of PML-NBs. Accumulating evidences have encouraged that PML-NBs represent preferential targets for viral infections and that PML plays an important role in the mechanism of the host intrinsic antiviral defenses. However, most of these studies mainly focus on hu-man viruses instead of the viruses infecting other animals. To date, the mechanism of PML antiviral defense is still unclear and the antiviral activity of swine PML (SPML) have not been reported. There-fore, studies on antiviral effect of swine PML will enhance the understanding of biological function and molecular mechanisms of PML in antiviral defense.We cloned different swine PML isoforms (I-V), analyzed their subcellular localization, and gener-ated the specific polyclonal antibody against SPML. Then, taking porcine pseudorabies virus (PRV) infection as a model, we investigated the potential antiviral activities of SPML; and screened the PRV proteins affecting the morphology and functions of SPML, and further explored the molecular mecha-nism responsible.PRV caused a rapid destruction of swine PML-NBs. To further investigate the viral genes involved in this process, we developed a simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9system. We show that up to100%viral gene disrupting efficiency was achieved by sim-ple cotransfection of the purified PRV genomes with CRISPR/Cas9system. Furthermore, CRISPR/Cas9-mediated knock-in of RFP cassette into the PRV genome at a positive rate of50%by a homology independent DNA repair mechanism withput constructing homology arms.The stable expression of SPMLII had slightly inhibitory effect on the proliferation of wild-type PRV, also owned significantly inhibitory effect on EPO null strain of PRV. We constructed a SPML"’" PK15cell line using TALEN tenology. In SPML+/+PK15cells, IFNa treatment significantly suppressed both wild-type and EPO mutant virus, but had a more obvious suppression on EPO mutant virus than that of wild-type virus. In SPML-/-PK15cells, however, the inhibitory effects of IFNa treatment on both wild-type and EPO mutant PRV became slight. Therefore, we concluded that the anti-PRV state induced by exogenous IFNa was enhanced by SPML and that the virus targeted the swine PML-NBs and dis-seminated SPML in order to preclude the establishment of the antiviral state induced by IFNa.PRV infection causes a dramatic disturbance of swine PML-NBs at early stage of infection. PRV early protein EPO can disrupt swine endogenous PML-NBs and the PML-NBs in PMLII-stable cell line. The disruption of PML-NBs mediated by PRV significantly decreased in EPO null PRV infection. PRV caused the degradation of stably expressed SPMLⅡ, which can be inhibited by proteasome inhibitors, while EPO null PRV virus lost its ability to degrade. These results suggested that the disruption of swine PML-NBs at the early stage of PRV infection was mediated by EPO.In sumary, we cloned and characterized swine PML; Swine PML had intrinsic antiviral effect and participated in the IFN-mediated immune defense response. We explored the mechanism by which PRV disrupted swine PML-NBs and PRV escaped swine PML antiviral effect. We developed the methods to manipulate PRV genome by using the CRISPR/Cas9system. These datas deepen our understandings of biological function and molecular mechanisms of PML in antiviral defense, which would provide im-portant theoretical basis and practical guidance for prevention and control of animal diseases. |
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