Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication - PubMed (original) (raw)
Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication
Chunchun Meng et al. Arch Virol. 2012 Jun.
Abstract
Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.
Figures
Fig. 1
NDV infection induces autophagy in U251 cells. (A) U251 cells were mock infected or infected with NDV at an MOI of 10 and prepared for TEM at 8 h postinfection. a, mock-infected cells showing normal distribution of organelles; b, NDV-infected cells appear highly vacuolated and show disappearance of most organelles; c, autophagic structures in virus-infected cells. The arrowhead shows cup-shaped membranous structures (phagophores, P) in the cytoplasm, wrapping around mitochondria. The arrow shows the two limiting membranes of an autophagosome, or initial autophagic vacuole (AVi). A vacuole containing membrane whorls, the late/degradative autophagic vacuole, is undergoing the degradation process; d, an autophagosome (AVi), containing a mitochondrion (M) and other organelles, is fusing with a lysosome (L) to form a single-membraned vacuole. The two limiting membranes (arrow) of the autophagosome are visible at the upper rim of the vacuole. (B) Formation of GFP-LC3 puncta in NDV-infected U251 cells. Cells were transfected with GFP-LC3, followed by NDV infection for 4 or 8 h. The cells with punctated GFP-LC3 were counted. Pictures from left to right show mock-infected cells, cells treated with rapamycin for 4 h as a positive control, and cells infected with NDV for 4 and 8 h, respectively. Cells with punctate GFP-LC3 were counted. **, p < 0.01. (C) and (D) The time of course induction of LC3II and expression of p62 in NDV-infected U251 cells were investigated by immunoblot (IB). R stands for rapamycin. Cells treated with rapamycin were used as the positive control. Sample loading was controlled using an anti-actin antibody as indicated. Densitometry was performed for quantification, and the ratios of LC3II to actin are presented below the blots. (E) U251 cells were infected with NDV at a range of MOIs (0.01, 0.1, 1 and 10) for 24 h, and LC3II production was measured by IB. (F) The time course of induction of LC3II in U251 cells treated with UV-inactivated NDV at an MOI of 10 was examined by IB. The results are representative of two separate experiments
Fig. 2
Signaling pathways involved in NDV infection of U251 cells contribute to virus-induced autophagy. U251 cells were infected with NDV at an MOI of 10 for different intervals as indicated in the figure. Cell lysates were analyzed for the activation of mTOR, p70S6K and Akt by immunoblot assay. The filters were stripped and reprobed with antibodies against total mTOR, p70S6K or Akt. Sample loading was controlled using an anti-actin antibody as indicated
Fig. 3
Regulation of NDV-induced autophagy in U251 cells affects virus production. (A) and (B) U251 cells were pretreated with rapamycin (Rapa) (100 nM) or chloroquine (CQ) (50 μM) and then infected with NDV (MOI = 0.01) at the indicated times, LC3II production was determined by immunoblot. The ratios of LC3II to actin are presented below the blots. (C) Virus yield in NDV-infected cells pretreated with or without rapamycin or chloroquine was determined at the indicated times. Data are presented as the mean ± SD for triplicate assays. **, p < 0.01
Fig. 4
Gene silencing of Atg5 or Beclin-1 by siRNA inhibits NDV production. U251 cells were transfected with 100 pmol siRNA duplexes targeted to either Atg5 or Beclin-1 mRNA, or control siRNA, for 72 h. (A) The relative expression of Atg5 and Beclin-1 proteins was examined by immunoblot analysis with specific antibody to Atg5 or Beclin-1, respectively. (B) Transfected cells were infected with NDV (MOI = 0.01) and the virus yield was determined at different intervals. Data are presented as the mean ± SD for triplicate assays. **, p < 0.01
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