RESUMEN
Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.
Asunto(s)
Conexina 43 , Homólogo 1 de la Proteína Discs Large , Queratinocitos , Humanos , Comunicación Celular , Membrana Celular/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Guanilato-Quinasas/metabolismo , Queratinocitos/metabolismo , Homólogo 1 de la Proteína Discs Large/genética , Homólogo 1 de la Proteína Discs Large/metabolismoRESUMEN
Regulation of human papillomavirus (HPV) gene expression is tightly linked to differentiation of the keratinocytes the virus infects. HPV late gene expression is confined to the cells in the upper layers of the epithelium where the virus capsid proteins are synthesized. As these proteins are highly immunogenic, and the upper epithelium is an immune-privileged site, this spatial restriction aids immune evasion. Many decades of work have contributed to the current understanding of how this restriction occurs at a molecular level. This review will examine what is known about late gene expression in HPV-infected lesions and will dissect the intricacies of late gene regulation. Future directions for novel antiviral approaches will be highlighted.
Asunto(s)
Virus del Papiloma Humano , Infecciones por Papillomavirus , Humanos , Animales , Papillomavirus Humano 16/genética , Diferenciación Celular , Queratinocitos/metabolismo , Queratinocitos/patología , Estadios del Ciclo de Vida , Papillomaviridae/genética , Replicación Viral/fisiologíaRESUMEN
The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air-liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.
Asunto(s)
Células Epiteliales/inmunología , Calor , Inmunidad Innata/inmunología , Interferones/inmunología , Mucosa Respiratoria/inmunología , SARS-CoV-2/inmunología , Replicación Viral/inmunología , Adolescente , Animales , COVID-19/genética , COVID-19/inmunología , COVID-19/virología , Chlorocebus aethiops , Células Epiteliales/metabolismo , Células Epiteliales/virología , Femenino , Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Interferones/genética , Interferones/metabolismo , Masculino , Persona de Mediana Edad , Modelos Biológicos , RNA-Seq/métodos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Técnicas de Cultivo de Tejidos , Células Vero , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
The infectious life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Evidence suggests a sophisticated interplay between host gene regulation and virus replication. Alternative splicing is an essential process for host and viral gene expression, and is generally upregulated by serine arginine-rich splicing factors (SRSFs). SRSF activity can be positively or negatively controlled by cycles of phosphorylation/dephosphorylation. Here we show that HPV16 infection leads to accumulation of the paradigm SRSF protein, SRSF1, in the cytoplasm in a keratinocyte differentiation-specific manner. Moreover, HPV16 infection leads to increased levels of cytoplasmic and nuclear phosphorylated SRSF1. SR protein kinase 1 (SRPK1) phosphorylates SRSF1. Similar to HPV upregulation of SRSF1, we demonstrate HPV upregulation of SRPK1 via the viral E2 protein. SRPK1 depletion or drug inhibition of SRPK1 kinase activity resulted in reduced levels of SRSF1, suggesting that phosphorylation stabilizes the protein in differentiated HPV-infected keratinocytes. Together, these data indicate HPV infection stimulates the SRPK1-SRSF axis in keratinocytes.
Asunto(s)
Empalme Alternativo/genética , Papillomavirus Humano 16/patogenicidad , Infecciones por Papillomavirus/genética , Proteínas Serina-Treonina Quinasas/genética , Células 3T3 , Animales , Células HeLa , Humanos , Queratinocitos/fisiología , Ratones , Fosforilación/genética , Factores de Empalme Serina-Arginina/genética , Regulación hacia Arriba/genética , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
Regulation of mRNA translation is a major control point for gene expression and is critical for life. Of central importance is the complex between cap-bound eukaryotic initiation factor 4E (eIF4E), eIF4G, and poly(A) tail-binding protein (PABP) that circularizes mRNAs, promoting translation and stability. This complex is often targeted to regulate overall translation rates, and also by mRNA-specific translational repressors. However, the mechanisms of mRNA-specific translational activation by RNA-binding proteins remain poorly understood. Here, we address this deficit, focusing on a herpes simplex virus-1 protein, ICP27. We reveal a direct interaction with PABP that is sufficient to promote PABP recruitment and necessary for ICP27-mediated activation. PABP binds several translation factors but is primarily considered to activate translation initiation as part of the PABP-eIF4G-eIF4E complex that stimulates the initial cap-binding step. Importantly, we find that ICP27-PABP forms a complex with, and requires the activity of, eIF4G. Surprisingly, ICP27-PABP-eIF4G complexes act independently of the effects of PABP-eIF4G on cap binding to promote small ribosomal subunit recruitment. Moreover, we find that a cellular mRNA-specific regulator, Deleted in Azoospermia-like (Dazl), also employs the PABP-eIF4G interaction in a similar manner. We propose a mechanism whereby diverse RNA-binding proteins directly recruit PABP, in a non-poly(A) tail-dependent manner, to stimulate the small subunit recruitment step. This strategy may be particularly relevant to biological conditions associated with hypoadenylated mRNAs (e.g., germ cells/neurons) and/or limiting cytoplasmic PABP (e.g., viral infection, cell stress). This mechanism adds significant insight into our knowledge of mRNA-specific translational activation and the function of the PABP-eIF4G complex in translation initiation.
Asunto(s)
Factor 4G Eucariótico de Iniciación/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismo , Animales , Factor 4G Eucariótico de Iniciación/genética , Femenino , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Modelos Biológicos , Mutación , Oocitos/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Unión a Poli(A)/genética , Unión Proteica , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
To assess the excess risk of HPV-associated cancer (HPVaC) in two at-risk groups-women with a previous diagnosis of high grade cervical intraepithelial neoplasia (CIN3) and both men and women treated for non-cervical pre-invasive anogenital disease. All CIN3 cases diagnosed in 1989-2015 in Scotland were extracted from the Scottish cancer registry (SMR06). All cases of pre-invasive penile, anal, vulval, and vaginal disease diagnosed in 1990-2015 were identified within the NHS pathology databases in the two largest NHS health boards in Scotland. Both were linked to SMR06 to extract subsequent incidence of HPVaC following the diagnosis of CIN3 or pre-invasive disease. Standardised incidence ratios were calculated for the risk of acquiring HPVaC for the two at-risk groups compared to the general Scottish population. Among 69,714 females in Scotland diagnosed with CIN3 (890,360.9 person-years), 179 developed non-cervical HPVaC. CIN3 cases were at 3.2-fold (95% CI: 2.7 to 3.7) increased risk of developing non-cervical HPVaC, compared to the general female population. Among 1,235 patients diagnosed with non-cervical pre-invasive disease (9,667.4 person-years), 47 developed HPVaC. Individuals with non-cervical pre-invasive disease had a substantially increased risk of developing HPVaC - 15.5-fold (95% CI: 11.1 to 21.1) increased risk for females and 28-fold (11.3 to 57.7) increased risk for males. We report a significant additional risk of HPV-associated cancer in those have been diagnosed with pre-invasive HPV-associated lesions including but not confined to the cervix. Uncovering the natural history of pre-invasive disease has potential for determining screening, prevention and treatment.
Asunto(s)
Neoplasias de los Genitales Femeninos/virología , Neoplasias de los Genitales Masculinos/virología , Infecciones por Papillomavirus/epidemiología , Lesiones Precancerosas/epidemiología , Displasia del Cuello del Útero/epidemiología , Neoplasias del Cuello Uterino/epidemiología , Adulto , Anciano , Canal Anal/patología , Femenino , Neoplasias de los Genitales Femeninos/epidemiología , Neoplasias de los Genitales Masculinos/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/complicaciones , Pene/patología , Estudios Retrospectivos , Escocia/epidemiología , Vagina/patología , Vulva/patologíaRESUMEN
Since their characterization more than five decades ago, gap junctions and their structural proteins-the connexins-have been associated with cancer cell growth. During that period, the accumulation of data and molecular knowledge about this association revealed an apparent contradictory relationship between them and cancer. It appeared that if gap junctions or connexins can down regulate cancer cell growth they can be also implied in the migration, invasion and metastatic dissemination of cancer cells. Interestingly, in all these situations, connexins seem to be involved through various mechanisms in which they can act either as gap-junctional intercellular communication mediators, modulators of signalling pathways through their interactome, or as hemichannels, which mediate autocrine/paracrine communication. This complex involvement of connexins in cancer progression is even more complicated by the fact that their hemichannel function may overlap with other gap junction-related proteins, the pannexins. Despite this complexity, the possible involvements of connexins and pannexins in cancer progression and the elucidation of the mechanisms they control may lead to use them as new targets to control cancer progression. In this review, the involvements of connexins and pannexins in these different topics (cancer cell growth, invasion/metastasis process, possible cancer therapeutic targets) are discussed.
Asunto(s)
Antineoplásicos/farmacología , Carcinogénesis , Conexinas/metabolismo , Metástasis de la Neoplasia , Neoplasias/metabolismo , Animales , Antineoplásicos/uso terapéutico , Comunicación Celular , Conexinas/antagonistas & inhibidores , Conexinas/fisiología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Transducción de SeñalRESUMEN
It is universally accepted that high-risk human papillomavirus (HR-HPV) is the cause of cervical dysplasia and cancer. More recently, it has been shown that HPV is also a marker of clinical outcome in oropharyngeal cancer. However, contemporary information is lacking on both the prevalence of HPV infection in vulvar cancer (VSCC), its precursor lesion, vulvar intraepithelial neoplasia (VIN) and the influence of HPV-status on the prognosis of this malignancy. We have conducted a detailed population-based study to examine rates of progression of VIN to VSCC, type-specific HPV prevalence in vulvar disease and the influence of HPV status on clinical outcome in VSCC. We observed that the age at which women are diagnosed with VSCC is falling and there is a significant time gap between first diagnosis of VIN and progression to invasive disease. HR-HPV infection was detected in 87% (97/112) cases of VIN and 52% cases (32/62) of VSCC. The presence of HR-HPV in squamous intraepithelial lesion was associated with lower rates of progression to invasive cancer (hazard ratio, 0.22, p = 0.001). In the adjusted analysis, HR-HPV was associated with improved progression-free survival of VSCC compared to those with HPV negative tumours (hazard ratio, 0.32, p = 0.02).
Asunto(s)
Carcinoma de Células Escamosas/virología , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Neoplasias de la Vulva/virología , Adulto , Anciano , Carcinoma in Situ/mortalidad , Carcinoma in Situ/terapia , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/terapia , ADN Viral/análisis , Bases de Datos Factuales , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Estimación de Kaplan-Meier , Liquen Escleroso y Atrófico/epidemiología , Liquen Escleroso y Atrófico/virología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/epidemiología , Papillomaviridae/genética , Infecciones por Papillomavirus/mortalidad , Infecciones por Papillomavirus/terapia , Prevalencia , Escocia/epidemiología , Fumar/epidemiología , Resultado del Tratamiento , Neoplasias de la Vulva/mortalidad , Neoplasias de la Vulva/terapiaRESUMEN
The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelium. This means that viral proteins must exert control over epithelial gene expression in order to optimize viral production. The HPV E2 protein controls replication, transcription, and viral genome partitioning during the viral infectious life cycle. It consists of a nucleic acid-binding domain and a protein-protein interaction domain separated by a flexible serine and arginine-rich hinge region. Over the last few years, mounting evidence has uncovered an important new role for E2 in viral and cellular RNA processing. This Gem discusses the role of E2 in controlling the epithelial cellular environment and how E2 might act to coordinate late events in the viral replication cycle.
Asunto(s)
Células Epiteliales/virología , Papillomaviridae/genética , Papillomaviridae/fisiología , Procesamiento Postranscripcional del ARN , Transcripción Genética , Proteínas Virales/metabolismo , Replicación Viral , HumanosRESUMEN
HPVs (human papillomaviruses) infect epithelial cells and their replication cycle is intimately linked to epithelial differentiation. There are over 200 different HPV genotypes identified to date and each displays a strict tissue specificity for infection. HPV infection can result in a range of benign lesions, for example verrucas on the feet, common warts on the hands, or genital warts. HPV infects dividing basal epithelial cells where its dsDNA episomal genome enters the nuclei. Upon basal cell division, an infected daughter cell begins the process of keratinocyte differentiation that triggers a tightly orchestrated pattern of viral gene expression to accomplish a productive infection. A subset of mucosal-infective HPVs, the so-called 'high risk' (HR) HPVs, cause cervical disease, categorized as low or high grade. Most individuals will experience transient HR-HPV infection during their lifetime but these infections will not progress to clinically significant cervical disease or cancer because the immune system eventually recognizes and clears the virus. Cancer progression is due to persistent infection with an HR-HPV. HR-HPV infection is the cause of >99.7% cervical cancers in women, and a subset of oropharyngeal cancers, predominantly in men. HPV16 (HR-HPV genotype 16) is the most prevalent worldwide and the major cause of HPV-associated cancers. At the molecular level, cancer progression is due to increased expression of the viral oncoproteins E6 and E7, which activate the cell cycle, inhibit apoptosis, and allow accumulation of DNA damage. This review aims to describe the productive life cycle of HPV and discuss the roles of the viral proteins in HPV replication. Routes to viral persistence and cancer progression are also discussed.
Asunto(s)
Neoplasias/virología , Papillomaviridae/fisiología , Infecciones por Papillomavirus/virología , Replicación Viral , División Celular , Femenino , Humanos , Masculino , Neoplasias/patología , Neoplasias/fisiopatología , Papillomaviridae/genética , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/fisiopatología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
UNLABELLED: High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. IMPORTANCE: Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.
Asunto(s)
Papillomavirus Humano 16/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Represoras/genética , Ribonucleoproteínas/metabolismo , Empalme Alternativo , Apoptosis , Línea Celular , Línea Celular Tumoral , Femenino , Regulación Viral de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Virales , Interacciones Huésped-Patógeno , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Ribonucleoproteínas/antagonistas & inhibidores , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virologíaRESUMEN
UNLABELLED: The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. We show here for the first time that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host serine-arginine (SR) proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export, and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and that HPV1 E1^E4 inhibits this phosphorylation. After mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following coexpression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4-mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts. IMPORTANCE: The HPV life cycle is tightly linked to the epithelial terminal differentiation program, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. We show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through the inhibition of a host cell kinase.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Interacciones Huésped-Patógeno , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transcripción Genética , Proteínas Virales/metabolismo , Línea Celular , Humanos , Fosforilación , Mapeo de Interacción de Proteínas , Replicación ViralRESUMEN
Molecular human papillomavirus (HPV) testing is an important and developing tool for cervical disease management. However there is a requirement to develop new HPV tests that can differentiate between clinically significant and benign, clinically insignificant infection. Evidence would indicate that clinically significant infection is linked to an abortive HPV replication cycle. In particular the later stages of the replication cycle (i.e., production of late messenger (m) RNAs and proteins) appear compromised. Compared to current DNA-based tests which indicate only presence or absence of virus, detecting virus mRNAs by reverse transcriptase PCR (RT-PCR) may give a more refined insight into viral activity and by implication, clinical relevance. A novel quantitative (q)RT-PCR assay was developed for the detection of mRNAs produced late in the viral replication cycle. Initially this was validated on HPV-containing cell lines before being applied to a panel of 223 clinical cervical samples representing the cervical disease spectrum (normal to high grade). Samples were also tested by a commercial assay which detects expression of early HPV E6/E7 oncoprotein mRNAs. Late mRNAs were found in samples associated with no, low and high grade disease and did not risk-stratify HPV infection. The data reveal hidden complexities within the virus replication cycle and associated lesion development. This suggests that future mRNA tests for cervical disease may require quantitative detection of specific novel viral mRNAs.
Asunto(s)
Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/diagnóstico , ARN Viral/genética , Enfermedades del Cuello del Útero/diagnóstico , Proteínas de la Cápside/genética , Línea Celular Transformada , Femenino , Genotipo , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Proteínas Represoras/genética , Enfermedades del Cuello del Útero/clasificaciónRESUMEN
Papillomavirus gene regulation is largely post-transcriptional due to overlapping open reading frames and the use of alternative polyadenylation and alternative splicing to produce the full suite of viral mRNAs. These processes are controlled by a wide range of cellular RNA binding proteins (RPBs), including constitutive splicing factors and cleavage and polyadenylation machinery, but also factors that regulate these processes, for example, SR and hnRNP proteins. Like cellular RNAs, papillomavirus RNAs have been shown to bind many such proteins. The life cycle of papillomaviruses is intimately linked to differentiation of the epithelial tissues the virus infects. For example, viral late mRNAs and proteins are expressed only in the most differentiated epithelial layers to avoid recognition by the host immune response. Papillomavirus genome replication is linked to the DNA damage response and viral chromatin conformation, processes which also link to RNA processing. Challenges with respect to elucidating how RBPs regulate the viral life cycle include consideration of the orchestrated spatial aspect of viral gene expression in an infected epithelium and the epigenetic nature of the viral episomal genome. This review discusses RBPs that control viral gene expression, and how the connectivity of various nuclear processes might contribute to viral mRNA production.
Asunto(s)
Regulación Viral de la Expresión Génica , Papillomaviridae , ARN Viral , Proteínas de Unión al ARN , Replicación Viral , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Humanos , ARN Viral/genética , ARN Viral/metabolismo , Papillomaviridae/genética , Papillomaviridae/fisiología , Proteínas Virales/metabolismo , Proteínas Virales/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/metabolismo , Genoma Viral , Interacciones Huésped-Patógeno , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Biobanks of cervical screening (LBC) samples annotated with disease status are an invaluable resource to support the development of tools for the risk stratification of disease. Although there is growing interest in the assessment of RNA-based biomarkers, little is known on the suitability and durability of stored clinical samples (commonly used in cervical screening) to support RNA-based research. RNA was extracted from 260 stored LBC samples. Storage at -80°C or -25°C allowed isolation of sufficient RNA for further analysis. RNA was found to be substantially degraded according to Agilent Bioanalyser data. Despite this, RT-qPCR was successful in 95% samples tested. These data suggest that biobanked LBC samples are suitable for RNA-based assessment even if stored for up to 14 years.
RNA was extracted from 260 cervical screening samples stored at either -80 or -25°C. An Agilent Bioanalyser was used to examine the level of degradation of a convenience sample of RNAs. Reverse transcriptase quantitative PCR (RT-qPCR) was used to quantify levels of two cellular mRNAs in all samples as a practical means of assessing suitability of the samples for mRNA biomarker analysis.
Asunto(s)
Manejo de Especímenes , Neoplasias del Cuello Uterino , Humanos , Femenino , Manejo de Especímenes/métodos , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/patología , ARN/análisis , ARN/aislamiento & purificación , ARN/genética , Cuello del Útero/citología , Detección Precoz del Cáncer/métodos , Bancos de Muestras Biológicas , Biomarcadores/análisis , Estabilidad del ARN , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , CitologíaRESUMEN
Human papillomaviruses (HPVs) infect cutaneous and mucosal epithelia to cause benign (warts) and malignant lesions (e.g. cervical cancer). Bovine papillomaviruses (BPVs) infect fibroblasts to cause fibropapillomas but can also infect cutaneous epithelial cells. For HPV-1, -16, -31 and BPV-1, cis-acting RNA elements in the late 3' untranslated region (3'UTR) control expression of virus proteins by binding host cell proteins. The present study compared the effects on gene expression of the cis-acting elements of seven PV late 3'UTRs (HPV-6b, -11, -16, -31 and BPV-1, -3 and -4) representing a range of different genera and species and pathological properties. pSV-beta-galactosidase reporter plasmids containing the late 3'UTRs from seven PVs were transiently transfected into cervical adenocarcinoma HeLa cells, and reporter gene expression quantified by reverse transcription-quantitative PCR and a beta-galactosidase assay. All elements inhibited gene expression in keratinocytes. Cancer-related types HPV-16 and -31, had the greatest inhibitory activity whereas the lowest inhibition was found in the non-cancer related types, BPV-3 and HPV-11. Using RBPmap version 1.1, bioinformatics predictions of factors binding the elements identified proteins which function mainly in mRNA splicing. Markedly, in terms of protein binding motifs, BPV late 3'UTR elements were similar to those of HPV-1a but not to other HPVs. Using HPV-1a as a model and siRNA depletion, the bioinformatics predictions were tested and it was found that PABPC4 was responsible for some of the 3'UTR repressive activity. The data revealed candidate proteins that could control PV late gene expression.
RESUMEN
The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners, including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C termini of ICP27. Expression of wild type ICP27 protein inhibited both classical, importin α/ß-dependent and transportin-dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling, thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways.
Asunto(s)
Transporte Activo de Núcleo Celular , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células HeLa , Herpesvirus Humano 8/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Inmunoprecipitación , Señales de Exportación Nuclear , Señales de Localización Nuclear , Poro Nuclear/virología , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Mapeo Peptídico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Poly(A)-binding protein 1 (PABP1) has a fundamental role in the regulation of mRNA translation and stability, both of which are crucial for a wide variety of cellular processes. Although generally a diffuse cytoplasmic protein, it can be found in discrete foci such as stress and neuronal granules. Mammals encode several additional cytoplasmic PABPs that remain poorly characterised, and with the exception of PABP4, appear to be restricted in their expression to a small number of cell types. We have found that PABP4, similarly to PABP1, is a diffusely cytoplasmic protein that can be localised to stress granules. However, UV exposure unexpectedly relocalised both proteins to the nucleus. Nuclear relocalisation of PABPs was accompanied by a reduction in protein synthesis but was not linked to apoptosis. In examining the mechanism of PABP relocalisation, we found that it was related to a change in the distribution of poly(A) RNA within cells. Further investigation revealed that this change in RNA distribution was not affected by PABP knockdown but that perturbations that block mRNA export recapitulate PABP relocalisation. Our results support a model in which nuclear export of PABPs is dependent on ongoing mRNA export, and that a block in this process following UV exposure leads to accumulation of cytoplasmic PABPs in the nucleus. These data also provide mechanistic insight into reports that transcriptional inhibitors and expression of certain viral proteins cause relocation of PABP to the nucleus.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Núcleo Celular/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Transporte de Proteínas/efectos de la radiación , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular , Animales , Apoptosis/efectos de la radiación , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Células HeLa , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Biosíntesis de Proteínas , Transporte de ARN , Proteínas Recombinantes/biosíntesis , Rayos UltravioletaRESUMEN
Persistent infection with cancer risk-related viruses leads to molecular, cellular and immune response changes in host organisms that in some cases direct cellular transformation. Alternative splicing is a conserved cellular process that increases the coding complexity of genomes at the pre-mRNA processing stage. Human and other animal tumour viruses use alternative splicing as a process to maximize their transcriptomes and proteomes. Medical therapeutics to clear persistent viral infections are still limited. However, specific lessons learned in some viruses [e.g. HIV and HCV (hepatitis C virus)] suggest that drug-directed inhibition of alternative splicing could be useful for this purpose. The present review describes the basic mechanisms of constitutive and alternative splicing in a cellular context and known splicing patterns and the mechanisms by which these might be achieved for the major human infective tumour viruses. The roles of splicing-related proteins expressed by these viruses in cellular and viral gene regulation are explored. Moreover, we discuss some currently available drugs targeting SR (serine/arginine-rich) proteins that are the main regulators of constitutive and alternative splicing, and their potential use in treatment for so-called persistent viral infections.
Asunto(s)
Empalme Alternativo , Regulación Viral de la Expresión Génica , Neoplasias/etiología , Neoplasias/terapia , Virus Oncogénicos/patogenicidad , Infecciones Tumorales por Virus , HumanosRESUMEN
Gap junctions, composed of Cxs (connexins), allow direct intercellular communication. Gap junctions are often lost during the development of malignancy, although the processes behind this are not fully understood. Cx43 is a widely expressed Cx with a long cytoplasmic C-terminal tail that contains several potential protein-interaction domains. Previously, in a model of cervical carcinogenesis, we showed that the loss of gap junctional communication correlated with relocalization of Cx43 to the cytoplasm late in tumorigenesis. In the present study, we demonstrate a similar pattern of altered expression for the hDlg (human discs large) MAGUK (membrane-associated guanylate kinase) family tumour suppressor protein in cervical tumour cells, with partial co-localization of Cx43 and hDlg in an endosomal/lysosomal compartment. Relocalization of these proteins is not due to a general disruption of cell membrane integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg interact directly in vitro and can form a complex in cells. This novel interaction requires the N- and C-termini of hDlg. hDlg is not required for Cx43 internalization in W12GPXY cells. Instead, hDlg appears to have a role in maintaining a cytoplasmic pool of Cx43. These results demonstrate that hDlg is a physiologically relevant regulator of Cx43 in transformed epithelial cells.