Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jul 20;416(1-2):9-15.
doi: 10.1016/j.virol.2011.04.017. Epub 2011 May 14.

Adenoviruses induce autophagy to promote virus replication and oncolysis

Humberto Rodriguez-Rocha  1 , Jorge G Gomez-Gutierrez, Aracely Garcia-Garcia, Xiao-Mei Rao, Lan Chen, Kelly M McMasters, Heshan Sam Zhou
Affiliations

Adenoviruses induce autophagy to promote virus replication and oncolysis

Humberto Rodriguez-Rocha et al. Virology. .

Abstract

Adenoviruses with deletion of E1b have been used in clinical trials to treat cancers that are resistant to conventional therapies. The efficacy of viral replication within cancer cells determines the results of oncolytic therapy, which remains poorly understood and requires further improvement. In this report, we show that adenoviruses induce autophagy by increasing the conversion of LC3-I to LC3-II and the formation of the Atg12-Atg5 complex. Inhibition of autophagy with 3-methyladenine (3MA) resulted in a decreased synthesis of adenovirus structural proteins, and thereby a poor viral replication; promotion of autophagy with rapamycin increased adenovirus yield. This study indicates that adenovirus-induced autophagy correlates positively with virus replication and oncolytic cell death, and that autophagy may generate nutrients that can be used for building viral progeny particles. These results further suggest that chemotherapeutic agents that increase cancer cell autophagy may improve the efficacy of oncolytic virotherapy.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Quantification of autophagy activation
A549 cells were transfected with pEAK12-Actin-LC3-dNGLUC followed by no infection (mock) or infection with Ad5, Adhz60 (MOI=4), or AdlacZ (MOI=10). AdlacZ with deletion of E1 gene is used as a negative control vector incapable of replication. Secretion of LC3/GLUC was monitored over the time with the Renilla Luciferase kit (Promega). Each point represents the mean of three independent experiments ± standard deviation (SD; bars).
Fig. 2
Fig. 2. Autophagy activation and integration of LC3 into autophagosome
A549 cells were transfected with pEGFP-LC3 followed by infection with Ad5, Adhz60, or AdlacZ. (A) Integration of GFP-LC3 into the autophagosome is depicted by punctate structures (indicated by arrows) and was analyzed by fluorescence microscopy at 36 h p.i. Images were taken with Kodak MDS 290 software (magnification ×40). (B) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. A representative experiment is shown from three performed.
Fig. 3
Fig. 3. Expression of the classical autophagy hallmarks
A549 cells were infected with Ad5, Adhz60, or AdLacZ. LC3 and Atg5 expression and modification were analyzed in a time-course assay. LC3-I and LC3-II were detected with a pAb anti-LC3. To detect Atg5 or Atg12-Atg5 complex, a pAb anti-Atg5 was used.
Fig. 4
Fig. 4. Effect of 3-methyladenine (3MA) on adenovirus yield and structural protein production
A549 cells were infected with Ad5 or Adhz60 in absence or presence of 3MA. Cells were harvested at indicated time points. (A) For titering, supernatants were serially diluted to determine the titers, plaque forming unit (PFU), in HEK-293 cells. Each point represents the mean of three independent experiments ± standard deviation (SD; bars; P < 0.05). (B) Ad structural proteins were detected with rabbit-antihuman Ad protein virions. One experiment is shown from three performed.
Fig. 5
Fig. 5. Inhibition or induction of autophagosome formation and inhibition of LC3-I to LC3-II conversion
(A) A549 cells were transfected with pEGFP-LC3 following infection with Ad5 (MOI=4) in absence or presence of 3MA (10 mM). (B) A549 cells were transfected with pEGFP-LC3 followed by no treatment or treatment with rapamycin (50 nM). Integration of GFP-LC3 into the autophagosome is depicted by punctate structures (indicated by arrows) and was analyzed by fluorescence microscopy at 36 h p.i. Images were taken with Kodak MDS 290 software (magnification, ×40). (C) A549 cells were infected with Ad5 in presence of 3MA (10 mM). LC3 expression was analyzed in a time-course assay. LC3-I and LC3-II were detected with a pAb anti-LC3. A representative experiment is shown from three performed.
Fig. 6
Fig. 6. Effect of pharmacological inhibition and stimulation of autophagy on adenovirus replication and oncolysis
(A) A549 cells were infected with Ad5 or Adhz60 in the presence or absence of 3MA (10 mM) or rapamycin (50 nM). Seventy-two h later, titers were determined by serial dilutions of supernatants and titers were measured by standard infection units. Cytotoxicity was analyzed by a MTT assay at 72 h after 3MA (B) or rapamycin (C) treatment. Each point represents the mean of three independent experiments ± (SD; bars).
Fig. 7
Fig. 7. Activation of autophagy in different types of cancer cells
Hela, HCT116, RKO, or H1299 cancer cell lines were transfected with pEAK12-Actin-LC3-dNGLUC followed by no infection (mock) or infection with Ad5 or Adhz60 (MOI=4). Secretion of LC3/GLUC was monitored at 36 h after infection with the Renilla Luciferase kit (Promega). Each point represents the mean of three independent experiments ± standard deviation (SD; bars).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baehrecke EH. Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol. 2005;6:505–510. - PubMed
    1. Baird SK, Aerts JL, Eddaoudi A, Lockley M, Lemoine NR, McNeish IA. Oncolytic adenoviral mutants induce a novel mode of programmed cell death in ovarian cancer. Oncogene. 2008;27:3081–3090. - PMC - PubMed
    1. Colombo MI. Pathogens and autophagy: subverting to survive. Cell Death Differ. 2005;12(Suppl 2):1481–1483. - PMC - PubMed
    1. Crompton AM, Kirn DH. From ONYX-015 to armed vaccinia viruses: the education and evolution of oncolytic virus development. Curr Cancer Drug Targets. 2007;7:133–139. - PubMed
    1. Garber K. China approves world’s first oncolytic virus therapy for cancer treatment. J Natl Cancer Inst. 2006;98:298–300. - PubMed

Publication types

MeSH terms