RESUMEN
Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpes Simple , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Animales , Ratones , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , Herpes Simple/virología , Herpes Simple/metabolismo , Humanos , Células Vero , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Chlorocebus aethiops , Replicación Viral , Virulencia , Línea Celular , Femenino , Ratones Endogámicos BALB C , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Transcripción GenéticaRESUMEN
Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.
Asunto(s)
Herpesvirus Humano 1 , Proteínas Virales , Replicación Viral , Animales , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/fisiología , Chlorocebus aethiops , Células Vero , Ratones , Proteínas Virales/genética , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Herpes Simple/virología , Virulencia , Femenino , HumanosRESUMEN
More than 100 different herpes simplex virus 1 (HSV-1) genes belong to three major classes, and their expression is coordinately regulated and sequentially ordered in a cascade. This complex HSV-1 gene expression is thought to be regulated by various viral and host cellular proteins. A host cellular protein, Myb-binding protein 1A (MYBBP1A), has been reported to be associated with HSV-1 viral genomes in conjunction with viral and cellular proteins critical for DNA replication, repair, and transcription within infected cells. However, the role(s) of MYBBP1A in HSV-1 infections remains unclear. In this study, we examined the effects of MYBBP1A depletion on HSV-1 infection and found that MYBBP1A depletion significantly reduced HSV-1 replication, as well as the accumulation of several viral proteins. These results suggest that MYBBP1A is an important host cellular factor that contributes to HSV-1 replication, plausibly by promoting viral gene expression.
Asunto(s)
Proteínas de Unión al ADN , Herpes Simple , Herpesvirus Humano 1 , Proteínas de Unión al ARN , Factores de Transcripción , Humanos , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Herpes Simple/virología , Herpesvirus Humano 1/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/genética , Proteínas Virales/farmacología , Replicación ViralRESUMEN
Cholangiocarcinoma (CCA) is a cancer with a poor prognosis due to difficulties in diagnosis and limited treatment options, highlighting the urgent need for new targeted therapies. In a clinical setting, we found that leukotriene levels in bile were higher than in serum. Immunohistochemical analysis of surgically resected samples also revealed that CysLT receptor 1 (CysLTR1) was more highly expressed in CCA than in normal bile duct tissue, prompting us to investigate leukotriene as a potential therapeutic target in CCA. In vitro studies using CCA cell lines expressing CysLTR1 showed that leukotriene D4, a major ligand of CysLTR1, promoted cell proliferation, with increased phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Additionally, treatment with two clinically available anti-allergic drugs-zileuton, an inhibitor of CysLT formation, and montelukast, a CysLTR1 inhibitor-had inhibitory effects on cell proliferation and migratory capacity, accompanied by the reduced phosphorylation of AKT and ERK1/2. Furthermore, the simultaneous administration of both drugs synergistically enhanced the inhibitory effect on cell proliferation. Our study suggests that use of these drugs may represent a novel approach to treat CCA through drug repositioning.
Asunto(s)
Neoplasias de los Conductos Biliares , Proliferación Celular , Colangiocarcinoma , Hidroxiurea , Antagonistas de Leucotrieno , Quinolinas , Receptores de Leucotrienos , Sulfuros , Humanos , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Proliferación Celular/efectos de los fármacos , Receptores de Leucotrienos/metabolismo , Antagonistas de Leucotrieno/farmacología , Antagonistas de Leucotrieno/uso terapéutico , Línea Celular Tumoral , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Sulfuros/farmacología , Quinolinas/farmacología , Hidroxiurea/análogos & derivados , Hidroxiurea/farmacología , Hidroxiurea/uso terapéutico , Acetatos/farmacología , Acetatos/química , Masculino , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Movimiento Celular/efectos de los fármacos , Femenino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Progresión de la Enfermedad , Leucotrienos/metabolismo , Fosforilación/efectos de los fármacos , Anciano , Leucotrieno D4/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacosRESUMEN
BACKGROUND: Despite advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to the lack of effective therapies. Our previous study showed that Luteolin (Lut), a flavonoid, suppressed pancreatocarcinogenesis and reduced the expression of dihydropyrimidine dehydrogenase (DPYD), an enzyme that degrades pyrimidines such as 5-fluorouracil (5-FU), in PDACs. In this study, we investigated the role of DPYD and evaluated the therapeutic potential of combining 5-FU with Lut in PDACs. METHODS AND RESULTS: PDAC cells overexpressing DPYD showed increased proliferation, and invasiveness, adding to the resistance to 5-FU. The xenograft tumors of DPYD-overexpressing PDAC cells also exhibit enhanced growth and invasion compared to the control xenograft tumors. RNA-seq analysis of the DPYD-overexpressing PDAC xenograft tumors revealed an upregulation of genes associated with metallopeptidase activity-MMP9 and MEP1A. Furthermore, the overexpression of MEP1A in PDAC was associated with invasion. Next, we investigated the combined effects of Lut, a DPYD suppressor, and 5-FU on DPYD-overexpressing xenograft tumors and PDAC of Pdx1-Cre; LSL-KrasG12D/+; Trp53flox/flox(KPPC) mice. Neither single administration of 5-FU nor Lut showed significant inhibitory effects; however, the combined administration of 5-FU and Lut exhibited a significant tumor-suppressive effect in both the xenograft tumors and KPPC models. CONCLUSION: We have elucidated that DPYD expression contributes to proliferation, invasiveness, and 5-FU resistance, in PDACs. The combination therapy of Lut and 5-FU holds the potential for enhanced efficacy against PDACs.