Browsing by Author "Jose, Leny"

Now showing 1 - 10 of 11
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    Dormant Mycobacterium tuberculosis converts isoniazid to the active drug in a Wayne’s model of dormancy
    (Nature, 2018) Raghunandanan, Sajith; Jose, Leny; Kumar, Ramakrishnan Ajay; Dermatology, School of Medicine
    Isoniazid (INH) is one among the four first-line drugs used in the treatment of tuberculosis. The bactericidal activity of INH is due to its ability to inhibit mycolic acid synthesis, which is an integral component of the mycobacterial cell wall. Non-replicating Mycobacterium tuberculosis (MTB) is phenotypically resistant to INH. The exact mechanism of this resistance is not clear, although the inability of dormant MTB to convert the pro-drug into an active form is thought to be one of the possible reasons. Employing targeted metabolomics approach, we show that dormant MTB can metabolize INH into its active INH-NAD+ adduct form. Further we show that the dormant bacteria have unaltered gene expression levels of katG and inhA (INH metabolizing enzymes). Transcript levels of drug efflux pump proteins which were low during dormancy did not increase in response to INH treatment. These findings point to an alternative mechanism for INH resistance in dormant MTB, which needs to be further elucidated.
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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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    Molecular advancements in the development of thermostable phytases
    (Springer, 2017-04) Rebello, Sharrel; Jose, Leny; Sindhu, Raveendran; Aneesh, Embalil Mathachan; Dermatology, School of Medicine
    Since the discovery of phytic acid in 1903 and phytase in 1907, extensive research has been carried out in the field of phytases, the phytic acid degradatory enzymes. Apart from forming backbone enzyme in the multimillion dollar-based feed industry, phytases extend a multifaceted role in animal nutrition, industries, human physiology, and agriculture. The utilization of phytases in industries is not effectively achieved most often due to the loss of its activity at high temperatures. The growing demand of thermostable phytases with high residual activity could be addressed by the combinatorial use of efficient phytase sources, protein engineering techniques, heterologous expression hosts, or thermoprotective coatings. The progress in phytase research can contribute to its economized production with a simultaneous reduction of various environmental problems such as eutrophication, greenhouse gas emission, and global warming. In the current review, we address the recent advances in the field of various natural as well as recombinant thermotolerant phytases, their significance, and the factors contributing to their thermotolerance.
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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 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 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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    Senataxin mediates R-loop resolution on HPV episomes
    (American Society for Microbiology, 2024) Jose, Leny; Smith, Keely; Crowner, Anaiya; Androphy, Elliot J.; DeSmet, Marsha; Dermatology, School of Medicine
    Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.