Targeting Human Papillomavirus Genome Replication for Antiviral Drug Discovery

Archambault, Jacques; Melendy, Thomas

doi:10.3851/imp2612

Cited by 25 publications

(20 citation statements)

References 113 publications

(163 reference statements)

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“…This is intriguing as previously the hypothesis was that E2 and Topo1 recruitment and stimulation were parallel and redundant mechanisms (Archambault & Melendy, 2013).…”

Section: Discussionmentioning

confidence: 82%

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Thomas

Androphy

2018

J Virol

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iii ACKNOWLEDGEMENTS I would like to thank my supervisor Elliot J. Androphy for his guidance, and encouragement over these many years. I truly appreciate your faith and continuous support.I also want to thank all members of the Androphy lab, for your guidance, critiques, and support.Thank you to my thesis committee, without whom none of this would be possible.Your guidance has been invaluable. Michael Klemsz, Suk-Hee Lee, Lindsey Mayo, and Andy Yu. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. v When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111. Human Papillomaviruses and DiseaseThere are over 200 HPVs that have been described and they manifest in a variety of ways; they can be asymptomatic, or they can result in benign lesions (papillomas) or progress to malignancy (de Villiers, Fauquet, Broker, Bernard, & zur Hausen, 2004). In the United States, HPV is the most common sexually transmitted infection and nearly all sexually active adults contract it at least once. E1The PV E1 protein is an essential viral replication factor (Lusky & Botchan, 1985;Ustav, Ustav, Szymanski, & Stenlund, 1991). E1 is the viral helicase and initiates replication by binding to specific sequences on the viral genome with the assistance of the E2 protein (Frattini & Laimins, 1994).There is sequence and functional homology between PV E1 and SV (simian virus) 40 Large T Antigen which allowed for many of the predictions concerning E2 shows differential affinity for the E2BS and it is proposed that at low levels, E2b...

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“…This is intriguing as previously the hypothesis was that E2 and Topo1 recruitment and stimulation were parallel and redundant mechanisms (Archambault & Melendy, 2013).…”

Section: Discussionmentioning

confidence: 82%

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Thomas

Androphy

2018

J Virol

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“…The major viral oncogenes E6 and E7 target, among other host proteins, p53 and pRb function, respectively, resulting in enhanced proliferation of the infected cell ( 3 ). Two viral proteins are required for replication of the viral genome: E1 and E2 ( 4 ). E2 is a DNA binding protein that forms homodimers and binds to 12-bp palindromic sequences in the LCR surrounding the A/T-rich origin of viral replication ( 5 ).…”

Section: Introductionmentioning

confidence: 99%

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

James

Das

Morgan

et al. 2020

mSphere

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Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle. IMPORTANCE HPV16 is the major human viral carcinogen, responsible for around 3 to 4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication, and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here, we describe the effects of WRN depletion on cellular differentiation with or without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle since replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.

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“…Topo1 has been previously reported to bind to both PVs E1 and E2 using bacterial protein expression and Southwestern blotting (14,15). These reports suggested that the loading of Topo1 was facilitated by E1, and a replication model was proposed where E1 interacts directly with Topo1 after E2 dissociation (16). However, there have been no in-cell experiments to corroborate these protein-protein interactions or the localization of Topo1 to an active PV ori.…”

Section: Resultsmentioning

confidence: 99%

Acetylation of E2 by P300 Mediates Topoisomerase Entry at the Papillomavirus Replicon

Thomas

Androphy

2019

J Virol

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Human papillomavirus (HPV) E2 proteins are integral for the transcription of viral genes and the replication and maintenance of viral genomes in host cells. E2 recruits the viral DNA helicase E1 to the origin. A lysine (K111), highly conserved among almost all papillomavirus (PV) E2 proteins, is a target for P300 (EP300) acetylation and is critical for viral DNA replication (E. , https://doi.org/10.1128/ JVI.01912-17). Since the viral genome exists as a covalently closed circle of doublestranded DNA, topoisomerase 1 (Topo1) is thought to be required for progression of the replication forks. Due to the specific effect of K111 mutations on DNA unwinding (Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI .01912-17), we demonstrate that the E2 protein targets Topo1 to the viral origin, and this depends on acetylation of K111. The effect was corroborated by functional replication assays, in which higher levels of P300, but not its homolog CBP, caused enhanced replication with wild-type E2 but not the acetylation-defective K111 arginine mutant. These data reveal a novel role for lysine acetylation during viral DNA replication by regulating topoisomerase recruitment to the replication origin. IMPORTANCE Human papillomaviruses affect an estimated 75% of the sexually active adult population in the United States, with 5.5 million new cases emerging every year. More than 200 HPV genotypes have been identified; a subset of them are linked to the development of cancers from these epithelial infections. Specific antiviral medical treatments for infected individuals are not available. This project examines the mechanisms that control viral genome replication and may allow the development of novel therapeutics.

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Targeting Human Papillomavirus Genome Replication for Antiviral Drug Discovery

Cited by 25 publications

References 113 publications

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Human Papillomavirus Replication Regulation by Acetylation of a Conserved Lysine in the E2 Protein

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

Acetylation of E2 by P300 Mediates Topoisomerase Entry at the Papillomavirus Replicon

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