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Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription.

Publication ,  Journal Article
Madigan, VJ; Yuziuk, JA; Chiarella, AM; Tyson, TO; Meganck, RM; Elmore, ZC; Tse, LV; Hathaway, NA; Asokan, A
Published in: PLoS Pathog
August 2019

Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears essential. Inhibition of ubiquitin-proteasome pathways revealed that blocking Valosin Containing Protein (VCP/p97), which targets substrates to the proteasome, can selectively and completely restore AAV-mediated transgene expression in RNF121 KO cells. Expanding on this finding, transcriptomic and proteomic analysis revealed that the catalytic subunit of DNA PK (DNAPK-Cs), a known activator of VCP, is upregulated in RNF121 KO cells and that the DNA damage machinery is enriched at sites of stalled AAV genome transcription. We postulate that a network of RNF121, VCP and DNA damage response elements function together to regulate transcriptional silencing and/or activation of AAV vector genomes.

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Published In

PLoS Pathog

DOI

EISSN

1553-7374

Publication Date

August 2019

Volume

15

Issue

8

Start / End Page

e1007988

Location

United States

Related Subject Headings

  • Virus Internalization
  • Virology
  • Valosin Containing Protein
  • Ubiquitination
  • Ubiquitin
  • Tumor Cells, Cultured
  • Transduction, Genetic
  • Transcriptome
  • Proteome
  • Membrane Proteins
 

Citation

APA
Chicago
ICMJE
MLA
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Madigan, V. J., Yuziuk, J. A., Chiarella, A. M., Tyson, T. O., Meganck, R. M., Elmore, Z. C., … Asokan, A. (2019). Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription. PLoS Pathog, 15(8), e1007988. https://doi.org/10.1371/journal.ppat.1007988
Madigan, Victoria J., Julianne A. Yuziuk, Anna M. Chiarella, Tyne O. Tyson, Rita M. Meganck, Zachary C. Elmore, Longping V. Tse, Nathaniel A. Hathaway, and Aravind Asokan. “Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription.PLoS Pathog 15, no. 8 (August 2019): e1007988. https://doi.org/10.1371/journal.ppat.1007988.
Madigan VJ, Yuziuk JA, Chiarella AM, Tyson TO, Meganck RM, Elmore ZC, et al. Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription. PLoS Pathog. 2019 Aug;15(8):e1007988.
Madigan, Victoria J., et al. “Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription.PLoS Pathog, vol. 15, no. 8, Aug. 2019, p. e1007988. Pubmed, doi:10.1371/journal.ppat.1007988.
Madigan VJ, Yuziuk JA, Chiarella AM, Tyson TO, Meganck RM, Elmore ZC, Tse LV, Hathaway NA, Asokan A. Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription. PLoS Pathog. 2019 Aug;15(8):e1007988.

Published In

PLoS Pathog

DOI

EISSN

1553-7374

Publication Date

August 2019

Volume

15

Issue

8

Start / End Page

e1007988

Location

United States

Related Subject Headings

  • Virus Internalization
  • Virology
  • Valosin Containing Protein
  • Ubiquitination
  • Ubiquitin
  • Tumor Cells, Cultured
  • Transduction, Genetic
  • Transcriptome
  • Proteome
  • Membrane Proteins