自噬作用能够增强猪瘟病毒在体外的复制

,The relationship between viral and autophagy,Method for detecting autophagy,自噬检测的金标准,透射电子显微镜是观察自噬现象的最直接、最经典的方法，电镜检测自噬主要是基于辨认自噬体结构。免疫金电镜技术来对电镜结果进行定量分析通过图像分析软件自动测量所有自噬囊泡的面积，在结合其他检测方法的基础上，如果一个细胞胞浆中被自噬性囊泡所占据的总面积增加，则可说明自噬机制的上调。,基于自噬体标记蛋白LC3 的检测方法,微管相关蛋白1轻链3(LC3)是自噬体膜上标记蛋 白细胞内存在两种形式的LC3蛋白：LC3和 LC3-LC3蛋白在合成后其C端即被Atg4蛋白 酶切割变成LC3-(细胞浆内)当自噬体形成后， LC3-和PE偶联形成LC3-(自噬体膜)LC3- 始终稳定地保留在自噬体膜上直到与溶酶体融合， LC3-的水平在某种程度上反映了自噬体的数量.,cell lines,PK-15 the model cell line for studying CSFV infection. 3D4/2 cell line representative of the macrophage that is the target for CSFV infection. Considering the noncytopathogenic nature of CSFVidentical multiplicities of infection (MOIs) and infection time points were used for both PK-15 and 3D4/2 cells.,CSFV infection increases the formation of autophagosome-like vesicles,(A) PK-15 and 3D4/2 cells were mock-infected or infected with CSFV at MOI of 1for48 h.White arrows indicate the struct ures of autophagosomes . LC3 protein immunogold labeling localized to the infection-associated membranes is indicated by black arrows in the infected cells. (B) Quantification of the autophagosome-like vesicles per cell image.,expression of autophagy marker proteins in CSFV-infected cells,PK-15 cells were mock-infected or infe cted with CSFV orUV- inactivated CSFV for 6, 12, 24, 36, and 48 h. the expression of LC3, ATG12ATG5, ATG5, BEDN1, E2, ACTB (loading control) were analyzed by immuno- blotting with specific antibodies . (B) 3D4/2 cells were infected and analyzed as in (A).,CSFV infection induces the redistribution of the autophagy marker LC3,(A)PK-15 cells were mock -infected or infected with CSFV or UV-inactivated CSFV or treated with rapamycin for 48 h. The cells were processed for indirect immunofluore- scence. The cell nucleus were counterstained with DAPI.The nucleus staining is shown in blue , E2 and NS5A staining is shown in green,LC3staining isshown in red , and the signals of colocalization are shown in yellow in merged image (B) 3D4/2 cells were infected and analyzed as in (A).,induction of autophagy with rapamycin enhances CSFV replication,PK-15 were pretreated with rapamycin or DMSO (control) for 1 h, followed by CSFV adsorption for 1. The cells were further cultured in fresh medium in the absence or presence of rapamycin . at 24 and 48 hpi, cell samples were analyzed by immunoblotting. The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to ACTB. The extracellular and intracellular copy numbers of CSFV were detected by qRT- PcR.,inhibition of autophagy with 3-MA reduces CSFV replication,PK-15 were pretreated with 3-MA or DMSO for4 h. After 1 h of virus absorption, the cells were further cultured in fresh medium in the absence or presence of 3MA.,inhibition of autophagy with LC3B-targeting shRNA reduces the replication of CSFV,PK-15 were transfected with shRNAs targeting LC3B or scrambled shRNAs for 48 h, followed by mock infection and CSFV infection.at 24 hpi, the silencing efficiency of LC3B shRNA, as well as the expression of autophagy marker proteins and CSFV-E2 were analyzed . .,Pharmacological alteration of autophagy does not affect cell viability,The cell viability of PK-15 and 3D4/2 (B) cells were determined by the MTT assay after treatments with rapamycin or 3-MA for 48 h.,In summary, they in vitro analysis demonstrates for the first time that CSFV needs to trigger a functional autophagy pathway to enhance virus replication and release in host cells. We postulate that autophagy may be a potential mechanism that CSFV uses to establish a persistent infection.