Human Papillomavirus 16 E2 Interaction with TopBP1 Is Required for E2 and Viral Genome Stability during the Viral Life Cycle

Prabhakar, Apurva T.; James, Claire D.; Fontan, Christian T.; Otoa, Raymonde; Wang, Xu; Bristol, Molly L.; Hill, Ronald D.; Dubey, Aanchal; Morgan, Iain M.

doi:10.1128/jvi.00063-23

Cited by 11 publications

(12 citation statements)

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“…This function of E2 promotes the nuclear localization of viral genomes following mitosis by allowing the viral genomes to “hitch-hike” onto the host mitotic chromatin using the E2-TopBP1 interaction. Disruption of the E2-TopBP1 interaction also resulted in a loss of E2 protein expression in HFK+HPV16 cells, promoting viral genome integration (20). In this report, we demonstrate that mutation of E2 K111 or K112 prevented the viral genome from immortalizing human foreskin keratinocytes (HFK, Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…We demonstrated that an interaction between HPV16 E2 (from now on E2 will mean HPV16 E2 unless stated otherwise) and TopBP1 is essential for E2 interaction with mitotic chromatin and E2 plasmid segregation function, and that the E2-TopBP1 interaction is dependent upon E2 phosphorylation on serine 23 by CK2 (20–22). More recently we demonstrated that an E2 interaction with both TopBP1 and BRD4 is required for E2 interaction with mitotic chromatin and E2 plasmid segregation function (23).…”

Section: Introductionmentioning

confidence: 85%

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A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels

Prabhakar,

James,

Youssef

et al. 2024

Preprint

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An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We also demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. The results present a model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis and E2 acetylation on K111 by p300 increases, promoting interaction with Top1 that protects K112 from ubiquitination and therefore E2 proteasomal degradation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 85%

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels

Prabhakar,

James,

Youssef

et al. 2024

Preprint

Self Cite

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“…One reason the virus activates the DDR is to promote HR replication of the viral genome, allowing amplification in the presence of an active DDR (22). A host of HR factors are recruited to the replicating viral genome and are critical for the viral life cycle (7, 8, 10, 12–14, 19, 44). Here we demonstrate that viral replication converts SAMHD1 into an HR factor via phosphorylation on threonine 592, the viral oncogenes E6 and E7 are unable to do this.…”

Section: Discussionmentioning

confidence: 99%

“…The process of viral replication activates the DNA damage response (DDR) and recruits a host of DNA repair factors to the viral DNA, including TopBP1, SIRT1 and WRN (7)(8)(9)(10)(11)(12)(13)(14)(15). Activation of the DDR is critical for the HPV life cycle, as is recruitment and interaction with host DNA repair factors, particularly those involved in homologous recombination (12,13,(16)(17)(18)(19)(20)(21). It is proposed that the recruitment of host DDR factors to the viral genome promote homologous recombination (HR) mediated DNA replication during the viral life cycle (22).…”

Section: Introductionmentioning

confidence: 99%

Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA

James,

Youssef,

Prabhakar

et al. 2023

Preprint

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We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the HPV16 life cycle. Here we demonstrate that in HPV negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phosphor-mimic (SAMHD1 T592D) reduces E1-E2 mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1 and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers.ImportanceHuman papillomaviruses are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here we demonstrate that HPV16 replication converts SAMHD1 into an HR factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16 immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16 immortalized human foreskin keratinocytes and HPV16 positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

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“…Phosphorylation of E2 by CK2 enhances its interaction with TOBP1, enabling the localization of the viral genome to chromatin sites that support viral replication during mitosis. Additionally, CK2 phosphorylation helps maintain the stabilization of E2 during cell differentiation 57–59 . The E2 protein binds to both the viral genome and host chromatin simultaneously, ensuring the retention of viral genomes in daughter nuclei upon the completion of mitosis 58 .…”

Section: Hpv and Its Pathogenesismentioning

confidence: 99%

Human papillomavirus associated cervical lesion: pathogenesis and therapeutic interventions

Ye,

Zheng,

et al. 2023

MedComm

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Human papillomavirus (HPV) is the most prevalent sexually transmitted virus globally. Persistent high‐risk HPV infection can result in cervical precancerous lesions and cervical cancer, with 70% of cervical cancer cases associated with high‐risk types HPV16 and 18. HPV infection imposes a significant financial and psychological burden. Therefore, studying methods to eradicate HPV infection and halt the progression of precancerous lesions remains crucial. This review comprehensively explores the mechanisms underlying HPV‐related cervical lesions, including the viral life cycle, immune factors, epithelial cell malignant transformation, and host and environmental contributing factors. Additionally, we provide a comprehensive overview of treatment methods for HPV‐related cervical precancerous lesions and cervical cancer. Our focus is on immunotherapy, encompassing HPV therapeutic vaccines, immune checkpoint inhibitors, and advanced adoptive T cell therapy. Furthermore, we summarize the commonly employed drugs and other nonsurgical treatments currently utilized in clinical practice for managing HPV infection and associated cervical lesions. Gene editing technology is currently undergoing clinical research and, although not yet employed officially in clinical treatment of cervical lesions, numerous preclinical studies have substantiated its efficacy. Therefore, it holds promise as a precise treatment strategy for HPV‐related cervical lesions.

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Human Papillomavirus 16 E2 Interaction with TopBP1 Is Required for E2 and Viral Genome Stability during the Viral Life Cycle

Cited by 11 publications

References 60 publications

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels

A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels

Human Papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA

Human papillomavirus associated cervical lesion: pathogenesis and therapeutic interventions

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