RESUMEN
Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.
Asunto(s)
Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Animales , Chlorocebus aethiops , Humanos , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Señales de Exportación Nuclear , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Línea Celular , Células Vero , Replicación ViralRESUMEN
N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.
Asunto(s)
Herpesvirus Humano 1/fisiología , Metiltransferasas/metabolismo , ARN Mensajero/metabolismo , Replicación Viral/fisiología , Animales , Proteínas de Ciclo Celular , Línea Celular , Chlorocebus aethiops , Regulación Enzimológica de la Expresión Génica , Humanos , Metiltransferasas/genética , Interferencia de ARN , Procesamiento Postranscripcional del ARN , Factores de Empalme de ARN , ARN Mensajero/genética , RNA-Seq/métodos , Células VeroRESUMEN
N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.
Asunto(s)
Coronavirus Humano OC43/fisiología , Procesamiento Postranscripcional del ARN/genética , SARS-CoV-2/fisiología , Replicación Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Línea Celular , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Proteínas de la Nucleocápside , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal or explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such this model allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.
Asunto(s)
Técnicas de Cultivo de Célula , Herpesvirus Humano 1/fisiología , Neuronas , Activación Viral/fisiología , Latencia del Virus/fisiología , Animales , Células Cultivadas , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Ratas , Ratas Sprague-DawleyRESUMEN
Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.
Asunto(s)
Genes Virales/genética , Herpesvirus Humano 1/genética , Nanoporos , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Línea Celular , Células Cultivadas , Células Epiteliales/virología , Fibroblastos/virología , Genoma Viral/genética , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Humanos , Neuronas/citología , Neuronas/virología , ARN Viral/genética , Proteínas Virales/genéticaRESUMEN
In cervical cancer, the association between HPV infection and dysregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTOR pathway) places mTOR as an attractive therapeutic target. The failure of current treatment modalities in advanced stages of this cancer and drawbacks of already available mTOR inhibitors demand for novel drug candidates. In the present study we identified the presence of a mTOR inhibitor in an active fraction of the ethyl acetate extract of Streptomyces sp OA293. The metabolites(s) in the active fraction completely inhibited mTORC1 and thereby suppressed activation of both of its downstream targets, 4E-BP1 and P70S6k, in cervical cancer cells. In addition, it also stalled Akt activation via inhibition of mTORC2. The mechanism of mTOR inhibition detailed in our study overcomes significant drawbacks of well known mTOR inhibitors such as rapamycin and rapalogs. The active fraction induced autophagy and Bax mediated apoptosis suggesting that mTOR inhibition resulted in programmed cell death of cancer cells. The molecular weight determination of the components in active fraction confirmed the absence of any previously known natural mTOR inhibitor. This is the first report of complete mTOR complex inhibition by a product derived from microbial source.
Asunto(s)
Productos Biológicos/farmacología , Streptomyces/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Neoplasias del Cuello Uterino/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Streptomyces/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
DEPTOR is an endogenous inhibitor of mTOR complexes, de-regulated in cancers. The present study reveals a vital role for DEPTOR in survival of cervical squamous cell carcinoma (SCC). DEPTOR was found to be overexpressed in both cervical SCC cells and tissues and it's silencing in cervical SCC cells induced apoptosis, mainly by up-regulation of p38 MAPK and by inhibiting PI3K/AKT pathway via a feed-back inhibition from mTORC1-S6K. DEPTOR silencing resulted in reduced expression of the nitric oxide synthases iNOS and eNOS, as well as increased activation of ERK1/2 and p38 MAP kinases. Activation of AKT signaling by overexpression of constitutively active-AKT (CA-AKT) failed to overcome the apoptosis caused by DEPTOR silencing. Similarly pharmacological inhibition of ERK also failed to control apoptosis. However pharmacological inhibition of p38 MAPK rescued the cells from apoptosis, indicating the major role of p38 MAPK in cell death induced by DEPTOR silencing. DEPTOR was also found to regulate ERK1/2 in an AKT dependent manner. DEPTOR knockdown induced cell death in SiHa cells overexpressing the anti-apoptotic Bcl-2 and Bcl-xL, indicating strong survival role of DEPTOR in these cells. DEPTOR overexpression activated PI3K/AKT by relieving the negative feed-back inhibition from mTORC1-S6K. DEPTOR regulation was also observed to be independent of HPV E6/E7 oncoproteins, but it might be a molecular co-factor contributing to cervical carcinogenesis. In summary, DEPTOR is found to promote survival of cervical SCC cells and its reduction induced apoptosis via differential effects on PI3K/AKT and p38 MAPK and can be a potential target in cervical SCC.