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ABSTRACT
Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.
Abbreviations
| DMSO | = | dimethyl sulfoxide |
| EGFP | = | enhanced green fluorescent protein |
| ER | = | endoplasmic reticulum |
| FBS | = | fetal bovine serum |
| G | = | the glycoprotein |
| GAPDH | = | glyceraldehyde-3-phosphate dehydrogenase |
| gRNA | = | geneome RNA |
| hpi | = | hours postinfection |
| HRP | = | horseradish peroxidase |
| L | = | the RNA-dependent RNA polymerase |
| MAP1LC3/LC3 | = | microtubule-associated protein 1 light chain 3 |
| M | = | matrix protein |
| MMP | = | mitochondrial membrane potential |
| MOI | = | multiplicity of infection |
| mRFP | = | monomeric red fluorescent protein |
| MTOR | = | mechanistic target of rapamycin (serine/threonine kinase) |
| N | = | he nucleoprotein |
| NA | = | mouse neuroblastoma cell line |
| P | = | phosphoprotein |
| PBS | = | phosphate-buffered saline |
| qRT-PCR | = | quantitative reverse transcriptase polymerase chain reaction |
| RABV | = | rabies virus |
| RIPA | = | radio-immunoprecipitation assay |
| SD | = | standard deviation |
| SK | = | human neuroblastoma cell line |
| TEM | = | transmission electron microscopy |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We are grateful to Shile Huang (Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, USA) for invaluable experimental help and discussion.
Funding
This study was partially supported by the National Program on Key Research Project of China (No. 2016YFD0500400), National Nature Science Foundation of China (No. 31172322), Nature Science Foundation of Guangdong (No.2015A03031103), and Special Fund for Agro-Scientific Research in the Public Interest (No. 201103032).