Browsing by Subject "Replication"

Now showing 1 - 9 of 9
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    Effects of Caffeine, a DNA Damage Response Inhibitor, on Papillomavirus Genome Replication
    (MDPI, 2022-11-05) Kanginakudru, Sriramana; Gilson, Timra; Jose, Leny; Androphy, Elliot J.; Dermatology, School of Medicine
    Epidemiological studies have revealed that caffeinated coffee imparts a reduced risk of oropharyngeal cancer, of which human papillomavirus (HPV) is one of the causative agents. Caffeine is a known inhibitor of the DNA damage response (DDR) pathway. We sought to test the effects of caffeine on the early replication of the HPV31 virus. It has been reported that the inhibition of several factors necessary for the DDR during the differentiation-dependent stage of HPV block genome amplification, while the HPV genome maintenance replication was unaffected. We first studied the effects of caffeine in the earliest stages of viral infection. Using pseudo-virions (PsV) expressing an m-Cherry reporter gene and quasi-virions (QsV) containing HPV31 genomes to mediate the infection, we found no evidence that caffeine impeded the viral entry; however, the infected cells displayed a reduced HPV copy number. In contrast, caffeine exposure increased the copy number of HPV31 episomes in the transient transfection assays and in the CIN612E cells that stably maintain viral episomes. There was a concomitant increase in the steady state levels of the HPV31 E1 and E2 transcripts, along with increased E2 loading at the viral origin of replication (ori). These results suggest that the caffeine-mediated inhibition of the DDR reduces viral genome replication in the early stage of infection, in contrast to the maintenance stage, in which the inhibition of the DDR may lead to an increase in viral amplicon replication.
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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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    HPV replication regulation by acetylation of a conserved lysine in the E2 protein
    (2017-06-26) Thomas, Yanique Serge Gillana; Androphy, Elliot J.; Klemsz, Michael; Yu, Andy; Mayo, Lindsey; Lee, Suk-Hee
    Papillomaviruses (PVs) are non-enveloped DNA viruses that are the primary etiological agents of cervical and oropharyngeal cancers. Vaccines for H(human)PV have proven to be effective prophylactic treatments; however, there is no treatment available for those currently infected. To develop new therapies, we require a clear understanding of viral pathogenesis and regulation. 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. 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.
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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 papillomavirus E2 protein by posttranslational modification
    (2015-04-24) Culleton, Sara Poirier; Androphy, Elliot J.; Klemsz, Michael; Mayo, Lindsey; Nakshatri, Harikrishna; Sullivan, William J., Jr.
    Papillomaviruses (PVs) are small, double-stranded DNA viruses. Hundreds of species have evolved to replicate in mammals, birds, and reptiles. Approximately two hundred species are estimated to infect humans alone, and these human papillomaviruses (HPVs) cause diseases ranging from benign warts to anogenital and oropharyngeal cancers. While vaccination is effective at preventing the majority of these infections and their disease outcomes, there are no successful treatments for existing infections; thus, exploration of novel therapeutic targets is warranted. PVs control expression and function of their gene products through alternative splicing, alternate start codons, and post-translational modification (PTM). The viral E2 protein regulates transcription, replication, and genome maintenance in infected cells, and PTMs have been demonstrated for E2 proteins from multiple papillomavirus types. Serine phosphorylation events were reported to influence E2 stability, and our laboratory was the first to describe in vitro acetylation events with implications for E2 transcription function. Here we report confirmation of these acetylation events in vivo and additional data elucidating the role of these PTMs in viral transcription. Moreover, we present a novel phosphorylation site for bovine papillomavirus type 1 (BPV-1) E2 at tyrosine 102 (Y102). Using phospho-deficient and phospho-mimetic point mutants, we found that this site influences E2-mediated transcription and replication, and we hypothesize that phosphorylation at Y102 regulates these activities by interrupting the association of E2 with its binding partners. We also report interaction of BPV-1 E2 and HPV-31 E2 with different receptor tyrosine kinases (TKs), most notably members of the fibroblast growth factor receptor family. We hypothesize that Y102 phosphorylation by these receptors occurs early in infection to limit viral replication and gene expression. Further studies will cement the role of RTKs in PV biology and could reveal novel therapeutic strategies.
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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.
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    Testing for Replication Competent Lentivirus Associated with HIV1 Lentiviral Vectors
    (Elsevier, 2003-05-01) Sastry, Lakshmi; Xu, Yi; Johnson, Terry; Cornetta, Ken; Medical and Molecular Genetics, School of Medicine
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    Transparent, Open, and Reproducible Prevention Science
    (Springer, 2022) Grant, Sean; Wendt, Kathleen E.; Leadbeater, Bonnie J.; Supplee, Lauren H.; Mayo-Wilson, Evan; Gardner, Frances; Bradshaw, Catherine P.; Pediatrics, School of Medicine
    The field of prevention science aims to understand societal problems, identify effective interventions, and translate scientific evidence into policy and practice. There is growing interest among prevention scientists in the potential for transparency, openness, and reproducibility to facilitate this mission by providing opportunities to align scientific practice with scientific ideals, accelerate scientific discovery, and broaden access to scientific knowledge. The overarching goal of this manuscript is to serve as a primer introducing and providing an overview of open science for prevention researchers. In this paper, we discuss factors motivating interest in transparency and reproducibility, research practices associated with open science, and stakeholders engaged in and impacted by open science reform efforts. In addition, we discuss how and why different types of prevention research could incorporate open science practices, as well as ways that prevention science tools and methods could be leveraged to advance the wider open science movement. To promote further discussion, we conclude with potential reservations and challenges for the field of prevention science to address as it transitions to greater transparency, openness, and reproducibility. Throughout, we identify activities that aim to strengthen the reliability and efficiency of prevention science, facilitate access to its products and outputs, and promote collaborative and inclusive participation in research activities. By embracing principles of transparency, openness, and reproducibility, prevention science can better achieve its mission to advance evidence-based solutions to promote individual and collective well-being.