Abstract
Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.
Highlights
Autophagy is induced by stress, starvation, and viral infections; the role of Ribonuclease L (RNase L) in autophagy has not been investigated
To further confirm that RNase L enzyme activity is required for induction of autophagy, RNase L knock out (KO) mouse embryonic fibroblasts (MEFs) were reconstituted with WT RNase L, RNase L R667A mutant that lacks nuclease activity or vector alone and transfected with 2–5A
Direct activation of RNase L by 2–5A stimulates autophagy as indicated by increased conversion of Light Chain 3-I (LC3-I) to lipidated LC3-II, degradation of p62 and microscopic evaluation of GFP-LC3 puncta formation. siRNA silencing of endogenous RNase L or RNase L KO MEFs decrease autophagy significantly
Summary
Autophagy is induced by stress, starvation, and viral infections; the role of RNase L in autophagy has not been investigated. Significance: Our findings identify a novel role of RNase L in inducing autophagy affecting outcomes of viral infections. Several proteins associated with autophagy regulate host responses to viral infections. RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Mammalian autophagy is regulated through a core complex formation between Beclin, a B-cell lymphoma-2 (Bcl-2)2-interacting protein, and phosphatidylinositol-3-kinase class 3 (PI3KC3/ Vps34), which participates in nucleation of autophagosome [4, 5]. Autophagy has diverse outcomes in the pathogenesis of viral infections: it may function as an antiviral mechanism to promote elimination of viruses and induce adaptive response or it may enhance viral replication or exit from cells [14]. Tel.: 419-530-2135; Fax: 419-530-7737; E-mail: Malathi.Krishnamurthy@ utoledo.edu
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