Browsing by Author "DeSmet, Marsha"

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    Focal Adhesion Kinase Binds to the HPV E2 Protein to Regulate Initial Replication after Infection
    (MDPI, 2023-09-28) Jose, Leny; Gonzalez, Jessica; Kessinger, Emma; Androphy, Elliot J.; DeSmet, Marsha; Dermatology, School of Medicine
    Human papillomaviruses are small DNA tumor viruses that infect cutaneous and mucosal epithelia. The viral lifecycle is linked to the differentiation status of the epithelium. During initial viral infection, the genomes replicate at a low copy number but the mechanism(s) the virus uses to control the copy number during this stage is not known. In this study, we demonstrate that the tyrosine kinase focal adhesion kinase (FAK) binds to and phosphorylates the high-risk viral E2 protein, the key regulator of HPV replication. The depletion of FAK with a specific PROTAC had no effect on viral DNA content in keratinocytes that already maintain HPV-16 and HPV-31 episomes. In contrast, the depletion of FAK significantly increased HPV-16 DNA content in keratinocytes infected with HPV-16 quasiviruses. These data imply that FAK prevents the over-replication of the HPV genome after infection through the interaction and phosphorylation of the E2 protein.
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    Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance
    (American Society for Microbiology, 2020-07-30) Gilson, Timra; Culleton, Sara; Xie, Fang; DeSmet, Marsha; Androphy, Elliot J.; Dermatology, School of Medicine
    Several serine and threonine residues of the papillomavirus early E2 protein have been found to be phosphorylated. In contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one in HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this study, we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the nonphosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither binds the C terminus of Brd4, but in all other aspects the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in vitro assay, while HPV-31 Y102E binds E1 and was able to replicate, albeit at lower levels than wild type. To examine the effect of E2 mutations under more native-like infection conditions, a neomycin-selectable marker was inserted into L1/L2 of the HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days posttransfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines.IMPORTANCE Posttranslational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.
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    Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication
    (Elsevier, 2015-04) Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.; Department of Dermatology, IU School of Medicine
    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number.
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    Papillomavirus E2 protein is regulated by specific fibroblast growth factor receptors
    (Elsevier, 2018-08) DeSmet, Marsha; Kanginakudru, Sriramana; Jose, Leny; Xie, Fang; Gilson, Timra; Androphy, Elliot J.; Dermatology, School of Medicine
    The papillomavirus (PV) E2 protein activates transcription and replication by recruiting cellular proteins and the E1 DNA helicase to their binding sites in the viral genome. We recently demonstrated that phosphorylation of tyrosine 102 in the bovine papillomavirus (BPV-1) E2 protein restricts these activities and that fibroblast growth factor receptor-3 (FGFR3) tyrosine kinase binds PV E2. Expression of FGFR3 decreased viral replication with both wild-type and the phenylalanine substitution at position 102, inferring that another kinase targets Y102. Here we tested FGFR- 1, −2 and −4 for association with PV E2 proteins. FGFR2 but not FGFR1 or FGFR4 co-immunoprecipitated with BPV-1 E2. We found that FGFR2 suppressed replication but did not depend on phosphorylation of BPV-1 Y102. HPV-16 and −31 E2 interacted with FGFR1, −2, and −4. These results imply that the expression and activity of FGF receptors in epithelial cells can regulate the function of E2 in viral replication.
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    Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication
    (American Society for Microbiology, 2019-05-01) DeSmet, Marsha; Jose, Leny; Isaq, Nasro; Androphy, Elliot J.; Dermatology, School of Medicine
    The papillomavirus (PV) E2 protein coordinates viral transcription and genome replication. Following a strategy to identify amino acids in E2 that are posttranslationally modified, we reported that tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) complexes with and phosphorylates E2, which inhibits viral DNA replication. Here, we present several lines of evidence indicating that tyrosine (Y) 138 of HPV-31 E2 is a substrate of FGFR3. The active form of FGFR3 bound to and phosphorylated the region of amino acids (aa) 107 to 175 in HPV-31 E2. The E2 phenylalanine (F) mutant Y138F displayed reduced FGFR3-induced phosphotyrosine. A constitutive kinase-active FGFR3 inhibited wild-type (WT) E2-induced E1-dependent DNA replication, while the 138F mutant retained activity. The tyrosine to glutamic acid (E) mutant Y138E, which can mimic phosphotyrosine, failed to induce transient DNA replication, although it maintained the ability to bind and localize the viral DNA helicase E1 to the viral origin. The bromodomain-containing protein 4 (Brd4) binds to E2 and is necessary for initiation of viral DNA synthesis. Interestingly, the Y138E protein coimmunoprecipitated with full-length Brd4 but was defective for association with its C-terminal domain (CTD). These results imply that the activity of the FGFR3 kinase in the infected epithelial cell restricts the HPV replication program through phosphorylation of E2 at Y138, which interferes with E2 binding to the Brd4 CTD, and that this interaction is required for initiation of viral DNA synthesis.IMPORTANCE Human papillomaviruses (HPVs) are highly infectious pathogens that commonly infect the oropharynx and uterine cervix. The idea that posttranslational modifications of viral proteins coordinates viral genome replication is less explored. We recently discovered that fibroblast growth factor receptor 3 (FGFR3) phosphorylates the viral E2 protein. The current study demonstrates that FGFR3 phosphorylates E2 at tyrosine 138, which inhibits association with the C-terminal peptide of Brd4. This study illustrates a novel regulatory mechanism of virus-host interaction and provides insight into the role of Brd4 in viral replication.
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    Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions
    (American Society for Microbiology, 2017-01-03) Culleton, Sara P.; Kanginakudru, Sriramana; DeSmet, Marsha; Gilson, Timra; Xie, Fang; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Qi, Guihong; Wang, Mu; Androphy, Elliot J.; Microbiology and Immunology, School of Medicine
    Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-to-glutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection. IMPORTANCE Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.
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    Phosphorylation of the Human Papillomavirus E2 Protein at Tyrosine 138 Regulates Episomal Replication
    (American Society for Microbiology, 2020-07) Jose, Leny; Androphy, Elliot J.; DeSmet, Marsha; Dermatology, School of Medicine
    The papillomavirus (PV) E2 protein is a critical regulator of viral transcription and genome replication. We previously reported that tyrosine (Y) 138 of HPV-31 E2 is phosphorylated by the fibroblast growth factor receptor 3 (FGFR3) kinase. In this study, we generated quasiviruses containing G418-selectable HPV-31 genomes with phosphodeficient phenylalanine mutant E2 Y138F and phosphomimetic glutamic acid mutant Y138E. We observed significantly fewer early viral transcripts immediately after infection with these Y138 mutant genomes even though E2 occupancy at the viral origin was equivalent to that of wild-type E2. Keratinocytes infected with Y138F quasiviruses formed stable colonies, and the genomes were maintained as episomes, while those infected with Y138E quasiviruses did not. We previously reported that the HPV-31 E2 Y138 mutation to glutamic acid did not bind to the Brd4 C-terminal motif (CTM). Here, we demonstrate that HPV-16 E2 Y138E bound to full-length Brd4 but not to the Brd4 CTM. We conclude that association of E2 with the Brd4 CTM is necessary for viral genome replication and suggest that this interaction can be regulated by phosphorylation of E2 Y138. IMPORTANCE Papillomavirus (PV) is a double-stranded DNA tumor virus infecting the cutaneous and mucosal epithelium. The PV E2 protein associates with a number of cellular factors to mediate replication of the HPV genome. Fibroblast growth factor receptor 3 (FGFR3) regulates HPV replication through phosphorylation of tyrosine 138 in the HPV E2 protein. Employing a quasivirus infection model and selection for G418 resistant genomes, we demonstrated that Y138 is a critical residue for Brd4 association and that inability to complex with Brd4 does not support episomal replication.
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    Pyk2 Regulates Human Papillomavirus Replication by Tyrosine Phosphorylation of the E2 Protein
    (American Society for Microbiology, 2020-09-29) Jose, Leny; DeSmet, Marsha; Androphy, Elliot J.; Dermatology, School of Medicine
    The human papillomavirus (HPV) E2 protein is a key regulator of viral transcription and replication. In this study, we demonstrate that the nonreceptor tyrosine kinase Pyk2 phosphorylates tyrosine 131 in the E2 transactivation domain. Both depletion of Pyk2 and treatment with a Pyk2 kinase inhibitor increased viral DNA content in keratinocytes that maintain viral episomes. The tyrosine-to-glutamic acid (E) mutant Y131E, which may mimic phosphotyrosine, failed to stimulate transient DNA replication, and genomes with this mutation were unable to establish stable episomes in keratinocytes. Using coimmunoprecipitation assays, we demonstrate that the Y131E is defective for binding to the C-terminal motif (CTM) of Bromodomain-containing protein 4 (Brd4). These data imply that HPV replication depends on E2 Y131 interaction with the pTEFb binding domain of Brd4.IMPORTANCE Human papillomaviruses are the major causative agents of cervical, oral, and anal cancers. The present study demonstrates that the Pyk2 tyrosine kinase phosphorylates E2 at tyrosine 131, interfering with genome replication. We provide evidence that phosphorylation of E2 prevents binding to the Brd4-CTM. Our findings add to the understanding of molecular pathways utilized by the virus during its vegetative life cycle and offers insights into the host-virus interactome.
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    Regulation of R-Loops in DNA Tumor Viruses
    (MDPI, 2024-10-02) Crowner, Anaiya; Smith, Keely; DeSmet, Marsha; Dermatology, School of Medicine
    R-loops are triple-stranded nucleic acid structures that occur when newly synthesized single-stranded RNA anneals to duplex DNA upon the collision of replication forks with transcription complexes. These RNA-DNA hybrids facilitate several transcriptional processes in the cell and have been described extensively in the literature. Recently, evidence has emerged that R-loops are key regulators of DNA tumor virus transcription and the replication of their lifecycle. Studies have demonstrated that R-loops on the Human Papillomavirus (HPV) genome must be resolved to maintain genome maintenance and avoid viral integration, a hallmark of HPV cancers. For Epstein-Barr virus (EBV), R-loops are formed at the oriLyt to establish lytic replication. Structural maintenance of chromosome proteins 5/6 (SMC5/6) bind to these viral R-loops to repress EBV lytic replication. Most viruses in the herpesvirales order, such as KSHV, contain R-loop-forming sequences. In this perspective, we will describe the current, although limited, literature demonstrating the importance of RNA-DNA hybrids to regulate DNA virus transcription. We will also detail potential new areas of R-loop research and how these viruses can be used as tools to study the growing field of R-loops.
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    Regulation of the Human Papillomavirus Lifecyle through Post-Translational Modifications of the Viral E2 Protein
    (MDPI, 2021-06-23) Jose, Leny; Gilson, Timra; Androphy, Elliot J.; DeSmet, Marsha; Dermatology, School of Medicine
    The human papillomavirus (HPV) is a DNA tumor virus that infects cutaneous and mucosal epithelia where high-risk (HR) HPV infections lead to cervical, oropharyngeal, and anogenital cancers. Worldwide, nearly 5% of all cancers are caused by HR HPV. The viral E2 protein is essential for episomal replication throughout the viral lifecycle. The E2 protein is regulated by phosphorylation, acetylation, sumoylation, and ubiquitination. In this mini-review, we summarize the recent advancements made to identify post translational modifications within E2 and their ability to control viral replication.