266: TOP1α and TOP3β Differentially Regulate HPV31 Replication via R-loops and DNA Breaks

Vatsa A et al., Proc. Natl. Acad. Sci. U.S.A. 2026.123:e2526296123 - In HPV31-positive keratinocytes (CIN612), TOP1α and TOP3β are upregulated and required for viral transcription and replication, acting via distinct effects on R-loop accumulation and topoisomerase-mediated DNA breaks. Key terms: HPV31, TOP1α, TOP3β, R-loops, DNA breaks.

Study Highlights:
Using HPV31-positive CIN612 cells and primary HFK controls, the authors applied ChIP, RADAR, DRIP, alkaline COMET, RNA-seq, and shRNA knockdown to map topoisomerase binding and function. They found TOP1α and TOP3β, but not TOP3α, are elevated in HPV-positive cells and bind the viral URR, and that shRNA depletion of TOP1α or TOP3β reduced episomal viral DNA and early viral transcripts. Knockdown decreased DNA breaks (~50% reduction in COMET tail formation and reduced γH2AX) and altered R-loop levels differentially, with TOP1α depletion increasing viral R-loops by ~50% and TOP3β depletion causing >3-fold R-loop accumulation at viral and cellular loci. Transcriptome changes included reduced IL6/STAT3-AKT signaling after TOP1α loss and marked downregulation of EGR3 (>5-fold) after TOP3β loss, linking distinct mechanistic effects to impaired viral replication.

Conclusion:
TOP1α and TOP3β are differentially required for maintenance of HPV episomes and viral gene expression through distinct regulation of DNA breaks, R-loop dynamics, and specific host signaling pathways.

Music:
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Article title:
Differential roles of type I topoisomerases in regulating HPV pathogenesis

First author:
Vatsa A

Journal:
Proc. Natl. Acad. Sci. U.S.A. 2026.123:e2526296123

DOI:
10.1073/pnas.2526296123

Reference:
Vatsa A, Templeton CW, Laimins L. Differential roles of type I topoisomerases in regulating HPV pathogenesis. Proc. Natl. Acad. Sci. U.S.A. 2026.123:e2526296123. https://doi.org/10.1073/pnas.2526296123

License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) - https://creativecommons.org/licenses/by/4.0/

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QC:
This episode was checked against the original article PDF and publication metadata for the episode release published on 2026-01-21.

QC Scope:
- article metadata and core scientific claims from the narration
- excludes analogies, intro/outro, and music
- transcript coverage: Audited sections covering HPV biology and torsional stress, TOP1α/TOP3β (and TOP3α) roles, URR binding and episomal maintenance, knockdown effects on viral DNA and transcription, R-loop dynamics (DRIP), DNA breaks (COMET, γH2AX, TOP1cc/TOP3cc), DDR involvement, and host signaling changes (IL-6/STAT3-AKT, EGR3).
- transcript topics: HPV-induced torsional stress and topoisomerase roles; TOP1α, TOP3β, and TOP3α expression in HPV-positive cells; Binding of TOP1α and TOP3β to the HPV URR and episomal maintenance; shRNA knockdown effects on HPV genome levels and viral transcription; R-loop dynamics and DRIP analyses; DNA damage and TOP1cc/TOP3cc formation; γH2AX and COMET results

QC Summary:
- factual score: 10/10
- metadata score: 10/10
- supported core claims: 8
- claims flagged for review: 0
- metadata checks passed: 4
- metadata issues found: 0

Metadata Audited:
- article_doi
- article_title
- article_journal
- license

Factual Items Audited:
- TOP1α and TOP3β are elevated in HPV-positive cells and bound to the HPV genome URR, while TOP3α is not bound to URR and is not essential for replication/transcription.
- Knockdown of TOP1α or TOP3β reduces HPV episome DNA and early viral transcripts (E1, E2, E6, E7).
- TOP1α depletion increases viral R-loops; TOP3β depletion increases R-loops more dramatically (greater than 3-fold).
- TOP1α and TOP3β knockdown reduces DNA breaks (COMET tail moment ~50% reduction; γH2AX decreases).
- TOP1α and TOP3β depletion reduces TOP1cc/TOP3cc formation; R-loop recruitment factors (DHX9, POLR3H) are modulated accordingly.
- Expression of HPV oncoproteins E6 and E7 upregulates TOP1α and TOP3β.

QC result: Pass.