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Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the t...

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Autores principales: Balakrishnan, Balaji, Sen, Dwaipayan, Hareendran, Sangeetha, Roshini, Vaani, David, Sachin, Srivastava, Alok, Jayandharan, Giridhara R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540029/
https://www.ncbi.nlm.nih.gov/pubmed/23320106
http://dx.doi.org/10.1371/journal.pone.0053845
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author Balakrishnan, Balaji
Sen, Dwaipayan
Hareendran, Sangeetha
Roshini, Vaani
David, Sachin
Srivastava, Alok
Jayandharan, Giridhara R.
author_facet Balakrishnan, Balaji
Sen, Dwaipayan
Hareendran, Sangeetha
Roshini, Vaani
David, Sachin
Srivastava, Alok
Jayandharan, Giridhara R.
author_sort Balakrishnan, Balaji
collection PubMed
description The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.
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spelling pubmed-35400292013-01-14 Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors Balakrishnan, Balaji Sen, Dwaipayan Hareendran, Sangeetha Roshini, Vaani David, Sachin Srivastava, Alok Jayandharan, Giridhara R. PLoS One Research Article The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer. Public Library of Science 2013-01-08 /pmc/articles/PMC3540029/ /pubmed/23320106 http://dx.doi.org/10.1371/journal.pone.0053845 Text en © 2013 Balakrishnan et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Balakrishnan, Balaji
Sen, Dwaipayan
Hareendran, Sangeetha
Roshini, Vaani
David, Sachin
Srivastava, Alok
Jayandharan, Giridhara R.
Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors
title Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors
title_full Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors
title_fullStr Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors
title_full_unstemmed Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors
title_short Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors
title_sort activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540029/
https://www.ncbi.nlm.nih.gov/pubmed/23320106
http://dx.doi.org/10.1371/journal.pone.0053845
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