RESUMO
BACKGROUND: Penile cancer (PeC) is a highly morbid disease which is rising in certain settings including Scotland. A component of PeC is associated with Human Papillomavirus (HPV) although its influence on clinical outcomes is debatable as is whether the fraction attributable to HPV is increasing. METHODS: A total of 122 archived tissue samples derived from patients diagnosed with PeC between 2006-2015 were collated and tested for HPV DNA using molecular PCR. HPV positivity was determined for the overall population and by calendar year of diagnosis to determine any temporal trends. The influence of age, deprivation, smoking, tumour stage and tumour grade on likelihood of HPV positivity was determined by logistic regression. In addition, the influence of HPV status and the other clinical and demographics variables on all-cause death and death from PeC was assessed. RESULTS: HPV was detected in 43 % (95 % CI: 34-52) of penile cancers and the majority of infections were HPV 16. The HPV component of PeC did not increase over the time period (p for linear trend - 0.226). No demographic or clinical variables were associated with HPV positivity neither was HPV status associated with improved all-cause or cancer-specific survival during the follow up period. CONCLUSION: The rise in PeC in Scotland may not be attributable to a rise in HPV-associated cancer; this is consistent with oropharyngeal cancer (OPC) in the UK where there is an increase in both HPV positive and negative cancer. This work calls for a larger multi centre study to enable further detailed investigation into the implications of HPV infection in PeC.
Assuntos
Alphapapillomavirus , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Neoplasias Penianas , Humanos , Masculino , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Neoplasias Penianas/epidemiologia , Estudos Retrospectivos , Escócia/epidemiologiaRESUMO
BACKGROUND: Although HPV-positive oropharyngeal cancer (OPC) patients have improved prognosis compared to HPV negative patients; there remains an HPV-positive group who have poor outcomes. Biomarkers to stratify discrete patient outcomes are thus desirable. Our objective was to analyse viral load (VL) by droplet digital PCR (ddPCR), in HPV-positive patients with OPC on whom clinical outcome data were available. METHODS: In a cohort of patients that had previously tested HPV positive via conventional PCR, VL was determined using ddPCR assays for HPV16 L1 and E6 genes. VL was classed as "medium/high" if more than 5.57 copies or 8.68 copies of the HPV 16 L1 or E6 gene were detected respectively. Effect of VL on overall survival and hazard of death & disease progression was performed with adjustments made for sex, age, deprivation, smoking, alcohol consumption and stage. RESULTS: L1 VL ranged from 0.0014-304 gene copies per cell with a mean of 30.9; comparatively E6 VL ranged from 0.0012-356 copies per cell with a mean of 37.9. Univariate analysis showed those with a medium/high VL had a lower hazard of death; this was significant for L1 (pâ¯=â¯0.02) but not for E6 (pâ¯=â¯0.67). The ratio of E6 to L1 deviated from nâ¯=â¯1 in most samples but had no influence on clinical outcomes. CONCLUSIONS: HPV viral load may be informative for the further stratification of clinical outcomes in HPV positive OPC patients.
Assuntos
Proteínas Oncogênicas Virais , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogênicas Virais/genética , Reação em Cadeia da Polimerase , Carga ViralRESUMO
AIMS: Oropharyngeal cancer (OPC) is increasing on a global scale, including the component driven by high-risk human papillomavirus (HR-HPV); contemporary data that provides insight into the prognosis of this disease in addition to the fraction attributable to HR-HPV are essential to inform primary and secondary disease management strategies. MATERIALS AND METHODS: A population-based cohort of 235 patients diagnosed with OPC between 2013 and 2015 in Scotland was assessed for HPV status using molecular genotyping. Associations between HR-HPV status and key clinical and demographic variables were estimated using the Pearson chi-squared test. Rates of overall survival and progression-free survival were estimated and visualised using Kaplan-Meier curves. RESULTS: HPV DNA (largely HPV 16) was identified in 60% of cases. After adjustment for age, gender, deprivation, smoking, alcohol consumption and tumour stage, patients with HR-HPV-positive OPC had an 89% reduction in the risk of death (hazard ratio = 0.11, 95% confidence interval 0.05-0.25) and an 85% reduction in the risk of disease progression (hazard ratio = 0.15, 95% confidence interval 0.07-0.30). HPV positivity was not associated with age, deprivation or smoking status, whereas those who reported excess alcohol consumption were less likely to be positive for HR-HPV. CONCLUSIONS: The prevalence of HR-HPV-associated OPC is high in Scotland and strongly associated with dramatically improved clinical outcomes, including survival. Demographic/behavioural variables did not reliably predict HPV positivity in this cohort, which underlines the importance of laboratory confirmation. Finally, the dominance of HPV 16 in OPC indicates the significant impact of prophylactic immunisation on this disease.
Assuntos
Imunização/métodos , Neoplasias Orofaríngeas/diagnóstico , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/patologia , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalized keratinocytes (NIKS) and NIKS stably transfected with HPV16 episomal genomes (NIKS16) were compared using next-generation sequencing (RNA-Seq). HPV16 infection altered expression of 2,862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNA-Seq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change), 670 genes were downregulated and 296 genes were upregulated. HPV downregulated many genes involved in epithelial barrier function, which involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant CCL20, and proinflammatory cytokines interleukin 1α (IL-1α) and IL-1ß. However, the type I interferon regulator IRF1, kappa interferon (IFN-κ), and viral restriction factors (IFIT1, -2, -3, and -5, OASL, CD74, and RTP4) were upregulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions, and cell adhesion. Quantitative PCR (qRT-PCR) and Western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE HPV genome amplification and capsid formation take place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNA-Seq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3,000 genes were differentially expressed in keratinocytes due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into the virus-host interaction that is crucial for the production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.
Assuntos
Papillomavirus Humano 16/patogenicidade , Queratinócitos/citologia , Infecções por Papillomavirus/genética , Análise de Sequência de RNA/métodos , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/virologia , Células 3T3 , Animais , Diferenciação Celular , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Papillomavirus Humano 16/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/virologia , Camundongos , Neoplasias do Colo do Útero/genética , Replicação Viral , Displasia do Colo do Útero/genéticaRESUMO
UNLABELLED: The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell, suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously, we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV type 16 (HPV16) infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35), and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here, we show that the E2 proteins of HPV16 and HPV31 control the expression of SRSFs 1, 2, and 3 in a differentiation-dependent manner. E2 has the greatest transactivation effect on expression of SRSF3. Small interfering RNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together, these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. IMPORTANCE: Human papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study, we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4(^)L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus, we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and the production of new virions.
Assuntos
Proteínas do Capsídeo/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genética , Papillomaviridae/fisiologia , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Diferenciação Celular , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Papillomavirus Humano 31/genética , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Estágios do Ciclo de Vida/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Replicação Viral/genéticaRESUMO
PURPOSE: A significant number of patients return to optometric practice dissatisfied with their spectacles. An important question is whether any of these cases are preventable. There are several different clinical maxims that are used to modify the subjective refraction when determining the refractive prescription. These maxims aim to improve patient comfort and adaptation and thereby reduce patient dissatisfaction with new spectacles. They are not based on research evidence, but rather on expert opinion gained from clinical experience. The aim of this study was to retrospectively analyze a large number of case records of dissatisfied patients to assess the possible usefulness of the prescribing maxim "if it ain't broke, don't fix it." METHODS: Three hundred eighteen non-tolerance cases from a university-based Canadian optometric clinic were categorized by a focus group of optometrists. Three prescribing categories were defined and comprised cases in which application of the proposed maxim may have prevented the recheck eye examination; a more limited application of the maxim for one working distance may have been appropriate; and finally scenarios in which the maxim did not work in that the practitioner was judged to have initially followed the maxim, yet patient dissatisfaction was still reported. The remaining unallocated records comprised prescribing situations outside the scope of this study. RESULTS: Approximately 32% of non-tolerance cases were judged to have been preventable by use of the proposed maxim. Furthermore, an additional 10% reduction in recheck cases may have been possible by a more liberal interpretation of the maxim. Conversely, 4% of cases were deemed to comprise scenarios in which the maxim was followed yet the patient returned later to report problems with their spectacles. CONCLUSIONS: The prescribing maxim "if it ain't broke, don't fix it" appears to have a role in reducing recheck eye examinations and improving patient satisfaction with new spectacles.
Assuntos
Óculos , Optometria/métodos , Satisfação do Paciente , Prescrições/normas , Erros de Refração/terapia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Seguimentos , Humanos , Lactente , Pessoa de Meia-Idade , Ontário , Optometria/normas , Refração Ocular , Erros de Refração/fisiopatologia , Estudos Retrospectivos , Adulto JovemRESUMO
HPV-16 is the major causes of cervical cancer. Persistence of infection is a necessary event for progression of the infection to cancer. Among other factors, virus persistence is due the viral proteins fighting the immune response. HPV-16 E5 down-regulates MHC/HLA class I, which is much reduced on the cell surface and accumulates in the Golgi apparatus in cells expressing E5. This effect is observed also in W12 cells, which mimic early cervical intraepithelial progression to cervical cancer. The functional effect of MHC I down-regulation on human CD8 T cells is not known, because of the need for HLA-matched, HPV-specific T cells that recognise E5 expressing-cells. Here we employ a heterologous cell/MHC I system which uses mouse cells expressing both E5 and HLA-A2, and A2-restricted CTLs; we show that the E5-induced reduction of HLA-A2 has a functional impact by reducing recognition of E5 expressing cells by HPV specific CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/biossíntese , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/patogenicidade , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Citometria de Fluxo , Antígeno HLA-A2/imunologia , Humanos , Evasão da Resposta Imune , Imuno-Histoquímica , Camundongos , Proteínas Oncogênicas ViraisRESUMO
Infection of cells by herpes simplex virus type 1 (HSV-1) triggers host cell shutoff whereby mRNAs are degraded and cellular protein synthesis is diminished. However, virus protein translation continues because the translational apparatus in HSV-infected cells is maintained in an active state. Surprisingly, poly(A)-binding protein 1 (PABP1), a predominantly cytoplasmic protein that is required for efficient translation initiation, is partially relocated to the nucleus during HSV-1 infection. This relocalization occurred in a time-dependent manner with respect to virus infection. Since HSV-1 infection causes cell stress, we examined other cell stress inducers and found that oxidative stress similarly relocated PABP1. An examination of stress-induced kinases revealed similarities in HSV-1 infection and oxidative stress activation of JNK and p38 mitogen-activated protein (MAP) kinases. Importantly, PABP relocalization in infection was found to be independent of the viral protein ICP27. The depletion of PABP1 by small interfering RNA (siRNA) knockdown had no significant effect on viral replication or the expression of selected virus late proteins, suggesting that reduced levels of cytoplasmic PABP1 are tolerated during infection.
Assuntos
Núcleo Celular/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Replicação Viral , Animais , Proteínas de Transporte , Núcleo Celular/genética , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Células HeLa , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Proteínas Imediatamente Precoces/genética , Estresse Oxidativo , Proteína I de Ligação a Poli(A)/genética , Transporte Proteico , Células VeroRESUMO
Human papillomavirus (HPV) L1 and L2 capsid protein expression is restricted to the granular layer of infected, stratified epithelia and is regulated at least partly at post-transcriptional levels. For HPV16, a 79 nt late regulatory element (LRE) is involved in this control. Using W12 cells as a model for HPV16-infected differentiating cervical epithelial cells we show that HuR, a key cellular protein that controls mRNA stability, binds the LRE most efficiently in nuclear and cytoplasmic extracts of differentiated cells. Further, HuR binds the 3' U-rich portion of the LRE directly in vitro. Overexpression of HuR in undifferentiated W12 cells results in an increase in L1 mRNA and protein levels while siRNA knock-down of HuR in differentiated W12 cells depletes L1 expression. In differentiated cervical epithelial cells HuR may bind and stabilise L1 mRNAs aiding translation of L1 protein.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas do Capsídeo/biossíntese , Células Epiteliais/virologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Feminino , Dosagem de Genes , Inativação Gênica , Humanos , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismoRESUMO
HPV-16 (human papillomavirus type 16) is a small dsDNA (double-stranded DNA) virus which infects mucosal epithelial tissue of the cervix. Epithelial tissue is composed of a basal layer of cells, capable of division, and a number of suprabasal layers, wherein the cells become more differentiated the closer to the surface of the epithelium they become. Expression of viral proteins is dependent upon epithelial differentiation status, and, within the HPV-16 genome, several elements have been found which control expression both transcriptionally and post-transcriptionally. Expression of the highly immunogenic capsid proteins, L1 and L2, is restricted to only the most differentiated cells, where immune surveillance is limited. However, L1 and L2 transcripts can be detected in less differentiated cells, suggesting post-transcriptional mechanisms exist to prevent their expression in these cells. Indeed, a number of cis-acting RNA elements have been observed within the HPV-16 late region which may be involved in control of capsid gene expression. Mechanisms controlling HPV-16 capsid gene expression and the cellular RNA-processing factors involved will be the focus of this article.
Assuntos
Processamento Alternativo , Papillomavirus Humano 16/genética , Diferenciação Celular , DNA Viral/genética , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Humanos , RNA Viral/genéticaRESUMO
In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3' untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.
Assuntos
Proteínas Nucleares , Papillomaviridae/genética , Papillomaviridae/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Diferenciação Celular , Núcleo Celular/fisiologia , Citoplasma/fisiologia , Células Epiteliais/citologia , Células HeLa , Humanos , Substâncias Macromoleculares , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AF , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
African trypanosomes first express the variant surface glycoprotein (VSG) at the metacyclic stage in the tsetse fly vector, in preparation for transfer into the mammal. Metacyclic (M)VSGs comprise a specific VSG repertoire subset and their expression is regulated differently from that of bloodstream VSGs, involving exclusively transcriptional regulation during the life cycle. To identify basic structural and functional features that may be common to MVSG telomeric transcription units, we have characterized the anatomy and transcription of the telomere containing the ILTat 1.61 MVSG gene. This telomere contains pseudogenes of the ESAG1 and ESAG9 families found in bloodstream VSG transcription units. The 1.61 MVSG occupies a monocistronic transcription unit and is transcriptionally controlled through the life cycle. The 1.61, and also the 1.22, MVSG transcription initiation site sequences resemble eukaryotic initiator elements. Sequence comparison reveals that four out of five characterized MVSG expression sites have a conserved region 2.0-4.7 kb long upstream of the MVSG. In some cases, this region contains not only the transcription initiation site that we have observed to be active in fly-transmitted trypanosomes but also, upstream, another sequence, described elsewhere as a 'putative promoter' for the MVAT set of M/VSGs (Nagoshi YL, Alarcon CM, Donelson JE. A monocistronic transcript for a trypanosome variant surface glycoprotein, Mol Biochem Parasitol 1995;72:33-45). In fly-transmitted trypanosomes, the latter element is transcriptionally silent. Our analysis of the structure of MVSG telomeres suggests that metacyclic expression sites arose from bloodstream expression sites.
Assuntos
Variação Antigênica/genética , Regiões Promotoras Genéticas , Telômero/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Sequência de Bases , Regulação da Expressão Gênica , Dados de Sequência Molecular , Transcrição Gênica , Trypanosoma brucei brucei/crescimento & desenvolvimento , Glicoproteínas Variantes de Superfície de Trypanosoma/biossínteseRESUMO
In the mammalian bloodstream, African trypanosomes express the variant surface glycoprotein (VSG), continual switching of which allows evasion of the host immune response. Bloodstream VSG genes are transcribed from polycistronic bloodstream expression sites with promoters which are located 45-60 kb upstream. These promoters are not exclusively stage-regulated, being active in the insect midgut stage where VSG is not expressed. However, the metacyclic VSG (M-VSG) genes, a small subset activated when VSG synthesis begins in the metacyclic stage in the tsetse fly salivary glands, are transcriptionally activated specifically in that stage from promoters <3 kb upstream. Using deletion mapping and transient transfection, we show that the entire 1.22 M-VSG gene promoter region (171 bp) is required for full activity in metacyclic-derived trypanosomes. However, a subsidiary, bloodstream stage-specific activity is present in its 5' half which directs transcription initiation very close to the initiation site used in metacyclic-derived trypanosomes. Our results imply that the M-VSG gene promoter is longer and more complex than other VSG gene promoters.
Assuntos
Genes de Protozoários , Regiões Promotoras Genéticas , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/biossíntese , Sequência Conservada , Eletroporação , Genes Reporter , Mamíferos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Roedores , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Transfecção , Tripanossomíase Africana/sangue , Tripanossomíase Africana/transmissão , Moscas Tsé-Tsé/parasitologiaRESUMO
As the metacyclic trypanosome stage develops in the tsetse fly salivary glands, it initiates expression of variant surface glycoproteins (VSGs) and does so by each cell activating, at random, one from a small subset of metacyclic VSG (M-VSG) genes. Whereas differential activation of individual VSG genes in the bloodstream occurs as a function of time, to evade waves of antibody, it is believed that the aim in the metacyclic stage is simultaneously to generate population diversity. M-VSG genes are activated in their telomeric loci and belong to monocistronic transcription units, unlike all other known trypanosome protein-coding genes, which appear to be transcribed polycistronically. The promoters of these metacyclic expression sites (M-ESs) have the unique property, in this organism, of being switched on and off in a life-cycle stage specific pattern. We have found that the 1.22 M-ES promoter is regulated according to life cycle stage, differential control being exerted through different elements of the promoter and under the influence of its genomic locus. We have characterized in detail the telomeres containing the 1.22 and 1.61 M-ESs. Upstream of the M-ES is a possibly haploid, non-transcribed region with some degenerate sequences homologous with expression site associated genes (ESAGs) that occur in bloodstream VSG expression sites. Further upstream (respectively, 22 and 13 kb upstream of the 1.22 and 1.61 VSG genes) are alpha-amanitin sensitive transcription units that may be polycistrons and are transcribed in all examined life cycle stages. They contain a number of genes. The differences between metacyclic and bloodstream ESs may have important consequences for life cycle regulation, genetic stability, phenotype complexity and adaptability of the metacyclic stage as it infects different host species.
Assuntos
Regulação da Expressão Gênica , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Genes de Protozoários , Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida , Regiões Promotoras Genéticas , Tripanossomíase Africana/parasitologiaRESUMO
African trypanosomes evade the mammalian host immune response by antigenic variation, the continual switching of their variant surface glycoprotein (VSG) coat. VSG is first expressed at the metacyclic stage in the tsetse fly as a preadaptation to life in the mammalian bloodstream. In the metacyclic stage, a specific subset (<28; 1 to 2%) of VSG genes, located at the telomeres of the largest trypanosome chromosomes, are activated by a system very different from that used for bloodstream VSG genes. Previously we showed that a metacyclic VSG (M-VSG) gene promoter was subject to life cycle stage-specific control of transcription initiation, a situation unique in Kinetoplastida, where all other genes are regulated, at least partly, posttranscriptionally (S. V. Graham and J. D. Barry, Mol. Cell. Biol. 15:5945-5956, 1985). However, while nuclear run-on analysis had shown that the ILTat 1.22 M-VSG gene promoter was transcriptionally silent in bloodstream trypanosomes, it was highly active when tested in bloodstream-form transient transfection. Reasoning that chromosomal context may contribute to repression of M-VSG gene expression, here we have integrated the 1.22 promoter, linked to a chloramphenicol acetyltransferase (CAT) reporter gene, back into its endogenous telomere or into a chromosomal internal position, the nontranscribed spacer region of ribosomal DNA, in both bloodstream and procyclic trypanosomes. Northern blot analysis and CAT activity assays show that in the bloodstream, the promoter is transcriptionally inactive at the telomere but highly active at the chromosome-internal position. In contrast, it is inactive in both locations in procyclic trypanosomes. Both promoter sequence and chromosomal location are implicated in life cycle stage-specific transcriptional regulation of M-VSG gene expression.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , CromossomosRESUMO
The major surface antigen of insect stage (procyclic and epimastigote form) Trypanosoma brucei is termed procyclin or procyclic acidic repetitive protein (PARP). Procyclin/PARP is not expressed in bloodstream form parasites, which are coated instead with the variant surface glycoprotein (VSG). Although procyclin/PARP protein is not present and the mRNA is barely detectable, procyclin/PARP genes are transcribed at this life cycle stage. We examined the mechanism for down-regulation of procyclin/PARP expression in bloodstream trypanosomes by using protein synthesis inhibitors to effect accumulation of procyclin/PARP transcripts. We show that the accumulation is not due to increased transcription of procyclin/PARP genes. Further, transcripts which accumulate under these conditions are of mature size, polyadenylated and polysome-associated indicating that normally, in bloodstream trypanosomes, down-regulation of procyclin/PARP expression is exerted either during transcript processing or at the level of mRNA stability. A comparison of the inhibitor-induced accumulation of procyclin/PARP transcripts in bloodstream forms of monomorphic and pleomorphic cell lines of trypanosome stock EATRO 795 shows that accumulation occurs with similar kinetics in both cell lines.
Assuntos
RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Cicloeximida/farmacologia , Expressão Gênica/efeitos dos fármacos , Cinética , Glicoproteínas de Membrana/genética , Camundongos , Polirribossomos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Protozoários/genética , Puromicina/farmacologia , RNA Mensageiro/genética , RNA de Protozoário/genética , Ratos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Regulação para Cima/efeitos dos fármacosRESUMO
The major surface antigen of procyclic and epimastigote forms of Trypanosoma congolense in the tsetse fly is GARP (glutamic acid/alanine-rich protein), which is thought to be the analogue of procyclin/PARP in Trypanosoma brucei. We have studied two T.congolense GARP loci (the 4.3 and 4.4 loci) whose transcription is alpha-amanitin sensitive. Whilst a transcriptional gap 5' of the first GARP gene in the cloned region of the 4.4 locus could not be detected, such a gap was present in the 5' flank of the first GARP gene in the 4.3 locus. We have located a GARP transcription start site and, using reporter gene constructs containing a putative GARP promoter region in transient transfection studies, we have demonstrated promoter activity for the test region in T.congolense. There are species-specific differences in sequences regulating expression of the two major surface antigens, GARP and procyclin/PARP: the GARP promoter is inactive in T.brucei while the procyclin/PARP promoter is inactive in T.congolense. We have defined the splice acceptor site for the 4.3 GARP gene by sequencing and by 5' RT-PCR and demonstrated microheterogeneity in GARP polyadenylation by 3' RT-PCR. It appears that some GARP and procyclin/PARP RNA processing signals, although similar, are also species-specific.
Assuntos
Amanitinas/farmacologia , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , Transcrição Gênica/efeitos dos fármacos , Trypanosoma congolense/genética , Animais , Antígenos de Protozoários/genética , Sequência de Bases , Regulação da Expressão Gênica , Genes de Protozoários/genética , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA de Protozoário/análise , RNA de Protozoário/metabolismo , Especificidade da Espécie , Trypanosoma brucei brucei/genética , Trypanosoma congolense/imunologiaRESUMO
In antigenic variation in African trypanosomes, switching of the variant surface glycoprotein (VSG) allows evasion of the mammalian host immune response. Trypanosomes first express the VSG in the tsetse fly vector, at the metacyclic stage, in preparation for transfer into the mammal. In this life cycle stage, a small, specific subset (1 to 2%) of VSGs are activated, and we have shown previously that the system of activation and expression of metacyclic VSG (M-VSG) genes is very different from that used for bloodstream VSG genes (S.V. Graham, K.R. Matthews, P.G. Shiels, and J.D. Barry, Parasitology 101:361-367, 1990). Now we show that unlike other trypanosome genes including bloodstream VSG genes, M-VSG genes are expressed from promoters subject to exclusively transcriptional regulation in a life cycle stage-dependent manner. We have located an M-VSG gene promoter, and we demonstrate that it is specifically up-regulated at the metacyclic stage. This is the first demonstration of gene expression being regulated entirely at the level of transcription among the Kinetoplastida; all other protein-coding genes examined in these organisms are, at least partly, under posttranscriptional control. The distinctive mode of expression of M-VSG genes may be due to a stochastic mechanism for metacyclic VSG activation.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Sequência de Bases , DNA de Protozoário/genética , Genes de Protozoários , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA de Protozoário/genética , Mapeamento por Restrição , Transcrição Gênica , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
During their life cycle, trypanosomatid parasites of mammals encounter substantially different environments in their hosts and insect vectors, to which they must adapt by undergoing a series of differentiation processes. At the molecular level, these processes must be the direct result of an elaborate series of changes in stage-regulated expression of a wide range of gene products. How are these changes accomplished? In this review, Sheila Graham discusses some recent advances in understanding the mechanisms of gene expression in trypanosomatids, and examines some clues to some intriguingly complex means of regulating life cycle stage-specific gene expression.