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

Victoria J Madigan,Tyne O Tyson,Anna M Chiarella,Zachary C Elmore,Julianne A Yuziuk,Longping V Tse,Rita M Meganck,Nathaniel A Hathaway,Aravind Asokan,Matthew D Weitzman

doi:10.1371/journal.ppat.1007988

Victoria J Madigan, Tyne O Tyson + Show 8 more

Open Access

https://doi.org/10.1371/journal.ppat.1007988

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Abstract

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.

Highlights

Adeno-associated viruses (AAVs) rely on co-infection of the host cell by a helper virus as well as several host factors for replication [1]
Recombinant AAV vectors are at the forefront of clinical gene therapy
We identify a network of host proteins involving ring finger protein 121 (RNF121), p97 and the DNA damage machinery as potent factors regulating AAV genome transcription

Summary

Introduction

Adeno-associated viruses (AAVs) rely on co-infection of the host cell by a helper virus as well as several host factors for replication [1]. The 4.7kb single stranded DNA AAV genome contains two open reading frames flanked by two inverted terminal repeats (ITRs) packaged into an icosahedral capsid measuring 25nm in diameter [1,2]. The only required cis-packaging signal for generating recombinant AAV vector genomes are the two ITRs [1,2]. AAV is thought to enter the nucleus through nuclear pores with the capsid intact, only uncoating once inside this environment. The uncoated ssDNA AAV genome undergoes second-strand synthesis and is transcribed, the impact of host factors on the latter event remains poorly understood [5,6]. Understanding post-second strand synthesis events in AAV biology could shed light on AAV vector genome silencing that has been observed in gene therapy clinical trials [6,7]

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