RESUMO
Human adenoviruses (HAdVs) are a large family of DNA viruses counting more than a hundred strains divided into seven species (A to G). HAdVs induce respiratory tract infections, gastroenteritis and conjunctivitis. APOBEC3B is a cytidine deaminase that restricts several DNA viruses. APOBEC3B is also implicated in numerous cancers where it is responsible for the introduction of clustered mutations into the cellular genome. In this study, we demonstrate that APOBEC3B is an adenovirus restriction factor acting through a deaminase-dependent mechanism. APOBEC3B introduces C-to-T clustered mutations into the adenovirus genome. APOBEC3B reduces the propagation of adenoviruses by limiting viral genome replication, progression to late phase, and production of infectious virions. APOBEC3B restriction efficiency varies between adenoviral strains, the A12 strain being more sensitive to APOBEC3B than the B3 or C2 strains. In A12-infected cells, APOBEC3B clusters in the viral replication centers. Importantly, we show that adenovirus infection leads to a reduction of the quantity and/or enzymatic activity of the APOBEC3B protein depending on the strains. The A12 strain seems less able to resist APOBEC3B than the B3 or C2 strains, a characteristic which could explain the strong depletion of the APOBEC3-targeted motifs in the A12 genome. These findings suggest that adenoviruses evolved different mechanisms to antagonize APOBEC3B. Elucidating these mechanisms could benefit the design of cancer treatments. This study also identifies adenoviruses as triggers of the APOBEC3B-mediated innate response. The involvement of certain adenoviral strains in the genesis of the APOBEC3 mutational signature observed in tumors deserves further study.
Assuntos
Infecções por Adenoviridae , Neoplasias , Humanos , Adenoviridae/genética , Adenoviridae/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Proteínas/metabolismo , Neoplasias/patologia , Antígenos de Histocompatibilidade Menor/genéticaRESUMO
Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.
Assuntos
Interferons , Mitocôndrias , Camundongos , Animais , Mitocôndrias/metabolismo , Vírus do Sarampo , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , DNA MitocondrialRESUMO
BACKGROUND: On May 6, 2022, a powerful outbreak of monkeypox virus (MPXV) had been reported outside of Africa, with many continuing new cases being reported around the world. Analysis of mutations among the 2 different lineages present in the 2021 and 2022 outbreaks revealed the presence of G->A mutations occurring in the 5'GpA context, indicative of APOBEC3 cytidine deaminase activity. METHODS: By using a sensitive polymerase chain reaction (differential DNA denaturation PCR) method allowing differential amplification of AT-rich DNA, we analyzed the level of APOBEC3-induced MPXV editing in infected cells and in patients. RESULTS: We demonstrate that G->A hypermutated MPXV genomes can be recovered experimentally from APOBEC3 transfection followed by MPXV infection. Here, among the 7 human APOBEC3 cytidine deaminases (A3A-A3C, A3DE, A3F-A3H), only APOBEC3F was capable of extensively deaminating cytidine residues in MPXV genomes. Hyperedited genomes were also recovered in â¼42% of analyzed patients. Moreover, we demonstrate that substantial repair of these mutations occurs. Upon selection, corrected G->A mutations escaping drift loss contribute to the MPXV evolution observed in the current epidemic. CONCLUSIONS: Stochastic or transient overexpression of the APOBEC3F gene exposes the MPXV genome to a broad spectrum of mutations that may be modeling the mutational landscape after multiple cycles of viral replication.
Assuntos
Citidina Desaminase , Monkeypox virus , Humanos , Monkeypox virus/genética , Citidina Desaminase/genética , Mutação , Surtos de Doenças , Citidina , Citosina Desaminase/química , Citosina Desaminase/genéticaRESUMO
Single-nucleotide polymorphism in APOBEC3C (resulting in a serine to isoleucine in position 188) is present in approximately 10% of African populations and greatly enhances restriction against human immunodeficiency virus-1 and simian immunodeficiency virus by improving dimerization and DNA processivity of the enzyme. In this study, we demonstrated in culture and in infected patients that hepatitis B virus (HBV) could be edited by APOBEC3CS188I. Using next-generation sequencing, we demonstrated that APOBEC3CS188I led to enhanced editing activity in 5'TpCpAâ5'TpTpA context. This constitutes a new hallmark of this enzyme, which could be used to determine its impact on HBV or nuclear DNA.
Assuntos
Citidina Desaminase , Genoma Viral , Vírus da Hepatite B , Citidina Desaminase/genética , Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The human APOBEC3A (A3A) cytidine deaminase is a powerful DNA mutator enzyme recognized as a major source of somatic mutations in tumor cell genomes. However, there is a discrepancy between APOBEC3A mRNA levels after interferon stimulation in myeloid cells and A3A detection at the protein level. To understand this difference, we investigated the expression of two novel alternative "A3Alt" proteins encoded in the +1-shifted reading frame of the APOBEC3A gene. A3Alt-L and its shorter isoform A3Alt-S appear to be transmembrane proteins targeted to the mitochondrial compartment that induce membrane depolarization and apoptosis. Thus, the APOBEC3A gene represents a new example wherein a single gene encodes two proapoptotic proteins, A3A cytidine deaminases that target the genome and A3Alt proteins that target mitochondria.
Assuntos
Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Mitocôndrias/genética , Proteínas/genética , Proteínas/fisiologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Citidina Desaminase/metabolismo , DNA/genética , Mutação da Fase de Leitura/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Humanos , Mitocôndrias/metabolismo , Mutação/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Fases de Leitura/genéticaRESUMO
BACKGROUND: APOBEC1 (A1) enzymes are cytidine deaminases involved in RNA editing. In addition to this activity, a few A1 enzymes have been shown to be active on single stranded DNA. As two human ssDNA cytidine deaminases APOBEC3A (A3A), APOBEC3B (A3B) and related enzymes across the spectrum of placental mammals have been shown to introduce somatic mutations into nuclear DNA of cancer genomes, we explored the mutagenic threat of A1 cytidine deaminases to chromosomal DNA. RESULTS: Molecular cloning and expression of various A1 enzymes reveal that the cow, pig, dog, rabbit and mouse A1 have an intracellular ssDNA substrate specificity. However, among all the enzymes studied, mouse A1 appears to be singular, being able to introduce somatic mutations into nuclear DNA with a clear 5'TpC editing context, and to deaminate 5-methylcytidine substituted DNA which are characteristic features of the cancer related mammalian A3A and A3B enzymes. However, mouse A1 activity fails to elicit formation of double stranded DNA breaks, suggesting that mouse A1 possess an attenuated nuclear DNA mutator phenotype reminiscent of human A3B. CONCLUSIONS: At an experimental level mouse APOBEC1 is remarkable among 12 mammalian A1 enzymes in that it represents a source of somatic mutations in mouse genome, potentially fueling oncogenesis. While the order Rodentia is bereft of A3A and A3B like enzymes it seems that APOBEC1 may well substitute for it, albeit remaining much less active. This modifies the paradigm that APOBEC3 and AID enzymes are the sole endogenous mutator enzymes giving rise to off-target editing of mammalian genomes.
Assuntos
Desaminase APOBEC-1/metabolismo , Cromossomos de Mamíferos/genética , Mutação , Desaminase APOBEC-1/química , Desaminase APOBEC-1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Ativação Enzimática , Expressão Gênica , Camundongos , Filogenia , Edição de RNA , Especificidade por SubstratoRESUMO
Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and ß production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing CâU editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CGâTA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer.
Assuntos
Citidina Desaminase/biossíntese , Quebras de DNA de Cadeia Dupla , DNA Mitocondrial/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citosol/metabolismo , Proteína DEAD-box 58 , DNA Mitocondrial/química , Humanos , Interferon-alfa/biossíntese , Interferon beta/biossíntese , Interferon beta/fisiologia , Proteínas/genética , Proteínas/metabolismo , RNA Polimerase III/metabolismo , Receptores Imunológicos , Transcrição Gênica , Regulação para Cima , Uracila/metabolismoRESUMO
The human APOBEC3A and APOBEC3B genes (A3A and A3B) encode DNA mutator enzymes that deaminate cytidine and 5-methylcytidine residues in single-stranded DNA (ssDNA). They are important sources of mutations in many cancer genomes which show a preponderance of CG->TA transitions. Although both enzymes can hypermutate chromosomal DNA in an experimental setting, only A3A can induce double strand DNA breaks, even though the catalytic domains of A3B and A3A differ by only 9% at the protein level. Accordingly we sought the molecular basis underlying A3B attenuation through the generation of A3A-A3B chimeras and mutants. It transpires that the N-terminal domain facilitates A3B activity while a handful of substitutions in the catalytic C-terminal domain impacting ssDNA binding serve to attenuate A3B compared to A3A. Interestingly, functional attenuation is also observed for the rhesus monkey rhA3B enzyme compared to rhA3A indicating that this genotoxic dichotomy has been selected for and maintained for some 38 million years. Expression of all human ssDNA cytidine deaminase genes is absent in mature sperm indicating they contribute to somatic mutation and cancer but not human diversity.
Assuntos
Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , Animais , Linhagem Celular , Citidina Desaminase/química , Citidina Desaminase/metabolismo , Células HeLa , Humanos , Macaca mulatta , Antígenos de Histocompatibilidade Menor , Mutação , Fenótipo , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Codorniz , Edição de RNARESUMO
BACKGROUND: The replication of HBV involves the production of covalently closed circular DNA (cccDNA) from the HBV genome through the repair of virion relaxed circular DNA (rcDNA) in the virion. As cccDNA is the transcription template for HBV genomes, it needs to be eliminated from hepatocytes if the eradication of chronic HBV infection is to be achieved. PCR quantitation of cccDNA copy number is the technique of choice for evaluating the efficiency of treatment regimens. The PCR target commonly used to identify cccDNA spans the gapped region of rcDNA and is considered to accurately distinguish between cccDNA and rcDNA. There is however, a potentially confounding issue in that PCR can generate larger targets from collections of small DNA fragments, a phenomenon known as PCR recombination. RESULTS: The impact of PCR recombination towards the amplification of this cccDNA specific target was explored by mixing three marked, yet overlapping HBV DNA fragments. Thirteen of sixteen possible recombinants were identified by sequencing with frequencies ranging from 0.6 to 23%. To confirm this finding in vivo, HBV positive sera were treated with DNase I and submitted to quantitative real-time PCR. Under these conditions, it was possible to amplify the cccDNA specific segment without difficulty. As the virion contains uniquely rcDNA, amplification of the cccDNA target resulted from PCR recombination. CONCLUSIONS: PCR quantitation of cccDNA may be more difficult than hitherto thought. Current detection protocols need to be investigated so as to help in the management of chronic HBV infection.
Assuntos
DNA Circular/análise , DNA Viral/análise , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Reação em Cadeia da Polimerase em Tempo Real , DNA Circular/sangue , DNA Viral/sangue , DNA Viral/genética , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Humanos , Vírion/genética , Replicação ViralRESUMO
The human APOBEC3 gene cluster locus encodes polynucleotide cytidine deaminases. Although many act as viral restriction factors through mutation of single-stranded DNA, recent reports have shown that human APOBEC3A was capable of efficiently hypermutating nuclear DNA and inducing DNA breaks in genomic DNA. In addition, the enzyme was unique in efficiently deaminating 5-methylcytidine in single-stranded DNA. To appreciate the evolutionary relevance of these activities, we analyzed A3A-related enzymes from the rhesus and tamarin monkey, horse, sheep, dog, and panda. All proved to be orthologous to the human enzyme in all these activities revealing strong conservation more than 148 My. Hence, their singular role in DNA catabolism is a well-established mechanism probably outweighing any deleterious or pathological roles such as genomic instability and cancer formation.
Assuntos
Citidina Desaminase/metabolismo , Citidina/análogos & derivados , DNA/genética , Mamíferos/metabolismo , Sequência de Aminoácidos , Animais , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , Desaminação , Cães , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Mamíferos/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Filogenia , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Alinhamento de SequênciaRESUMO
BACKGROUND: The role of innate immunity in general and of type I interferon (IFN-I) in particular in HTLV-1 pathogenesis is still a matter of debate. ADAR1-p150 is an Interferon Stimulated Gene (ISG) induced by IFN-I that can edit viral RNAs. We therefore investigated whether it could play the role of an anti-HTLV factor. RESULTS: We demonstrate here that ADAR1 is also expressed in the absence of IFN stimulation in activated primary T-lymphocytes that are the natural target of this virus and in HTLV-1 or HTLV-2 chronically infected T-cells. ADAR1 expression is also increased in primary lymphocytes obtained from HTLV-1 infected individuals. We show that ADAR1 enhances HTLV-1 and HTLV-2 infection in T-lymphocytes and that this proviral effect is independent from its editing activity. ADAR1 expression suppresses IFN-α inhibitory effect on HTLV-1 and HTLV-2 and acts through the repression of PKR phosphorylation. DISCUSSION: This study demonstrates that two interferon stimulated genes, i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. The balanced expression of those proteins could determine the fate of the viral cycle in the course of infection.
Assuntos
Adenosina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , eIF-2 Quinase/antagonistas & inibidores , Células Cultivadas , Humanos , Inibição Psicológica , Dados de Sequência Molecular , Análise de Sequência de DNA , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
The human APOBEC3 (A3A-A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome.
Assuntos
Citosina Desaminase/metabolismo , DNA Mitocondrial/metabolismo , Loci Gênicos , Genoma Humano , Mutação , Desaminases APOBEC , Citidina Desaminase , Citosina Desaminase/genética , DNA Mitocondrial/genética , Feminino , Células HeLa , Hepadnaviridae/genética , Hepadnaviridae/metabolismo , Humanos , Masculino , Neoplasias/enzimologia , Neoplasias/genética , Retroviridae/genética , Retroviridae/metabolismo , Uracila-DNA Glicosidase/deficiência , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
BACKGROUND: Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. METHODS: To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DESE439K mutation, and post-mortem heart samples from five control healthy donors. RESULTS: The heterozygous DESE439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient's heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. CONCLUSIONS: This work highlights the deleterious effects of DESE439K mutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.
Assuntos
Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Humanos , Desmina/genética , Desmina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Cardiomiopatias/metabolismo , Mutação/genética , Miócitos Cardíacos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.
Assuntos
Núcleo Celular/metabolismo , Citidina Desaminase/metabolismo , Citidina/química , DNA/metabolismo , Regulação da Expressão Gênica , Proteínas/metabolismo , Ciclo Celular , Sobrevivência Celular , Citidina Desaminase/fisiologia , Reparo do DNA , Desaminação , Genoma , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal/métodos , Mutagênese , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Recombination is an important mechanism in the generation of genetic diversity of the human (HIV) and simian (SIV) immunodeficiency viruses. It requires the co-packaging of divergent RNA genomes into the same retroviral capsid and subsequent template switching during the reverse transcription reaction. By HIV-specific fluorescence in situ hybridization (FISH), we have previously shown that the splenocytes from 2 chronically infected patients with Castelman's disease were multi-infected and thus fulfill the in vivo requirements to generate genetic diversity by recombination. In order to analyze when multi-infection first occurs during a lentivirus infection and how the distribution of multi-infection evolves during the disease course, we now determined the SIV copy numbers from splenocytes of 11 SIVmac251-infected rhesus macaques cross-sectionally covering the time span of primary infection throughout to end-stage immunodeficiency. RESULTS: SIV multi-infection of single splenocytes was readily detected in all monkeys and all stages of the infection. Single-infected cells were more frequent than double- or triple- infected cells. There was no strong trend linking the copy number distribution to plasma viral load, disease stage, or CD4 cell counts. CONCLUSIONS: SIV multi-infection of single cells is already established during the primary infection phase thus enabling recombination to affect viral evolution in vivo throughout the disease course.
Assuntos
Recombinação Genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Baço/virologia , Superinfecção/virologia , Animais , Dosagem de Genes , Macaca mulatta , Provírus/genética , Baço/citologia , Integração ViralRESUMO
We sought to examine ADAR-1 editing of measles and influenza virus genomes derived from inactivated seasonal influenza and live attenuated measles virus vaccines grown on chicken cells as the culture substrate. Using highly sensitive 3DI-PCR (R. Suspène et al., Nucleic Acids Res. 36:e72, 2008), it was possible to show that ADAR-1 could hyperdeaminate adenosine residues in both measles virus and influenza virus A genomes. Detailed analysis of the dinucleotide editing context showed preferences for 5'ArA and 5'UrA, which is typical of editing in mammalian cells. The hyperedited mutant frequency, including genomes and antigenomes, was a log greater for influenza virus compared to measles virus, suggesting a greater sensitivity to restriction by ADAR-1.
Assuntos
Adenosina Desaminase/metabolismo , Genoma Viral , Vírus da Influenza A/genética , Influenza Humana/enzimologia , Vírus do Sarampo/genética , Sarampo/enzimologia , Mutação , Adenosina Desaminase/genética , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Humanos , Vacinas contra Influenza/genética , Influenza Humana/virologia , Sarampo/virologia , Vacina contra Sarampo/genética , Dados de Sequência Molecular , Proteínas de Ligação a RNA , Estações do Ano , Vacinas Atenuadas/genética , Células VeroRESUMO
Human APOBEC3 cytidine deaminases target and edit single-stranded DNA, which can be of viral, mitochondrial, or nuclear origin. Retrovirus genomes, such as human immunodeficiency virus (HIV) genomes deficient in the vif gene and the hepatitis B virus genome, are particularly vulnerable. The genomes of some DNA viruses, such as human papillomaviruses, can be edited in vivo and in transfection experiments. Accordingly, herpesviruses should be no exception. This is indeed the case for herpes simplex virus 1 (HSV-1) in tissue culture, where APOBEC3C (A3C) overexpression can reduce virus titers and the particle/PFU ratio â¼10-fold. Nonetheless, A3A, A3G, and AICDA can edit what is presumably a small fraction of HSV genomes in an experimental setting without seriously impacting the viral titer. Hyperediting was found in HSV genomes recovered from 4/8 uncultured buccal lesions. The phenomenon is not restricted to HSV, since hyperedited Epstein-Barr virus (EBV) genomes were readily recovered from 4/5 established cell lines, indicating that episomes are vulnerable to editing. These findings suggest that the widely expressed A3C cytidine deaminase can function as a restriction factor for some human herpesviruses. That the A3C gene is not induced by type I interferons begs the question whether some herpesviruses encode A3C antagonists.
Assuntos
Citosina Desaminase/metabolismo , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 4/genética , Desaminases APOBEC , Animais , Sequência de Bases , Chlorocebus aethiops , Citidina Desaminase , Citosina Desaminase/genética , DNA/genética , Células HeLa , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 4/fisiologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Células Vero , Replicação ViralRESUMO
DNA viruses, retroviruses and hepadnaviruses, such as hepatitis B virus (HBV), are vulnerable to genetic editing of single stranded DNA by host cell APOBEC3 (A3) cytidine deaminases. At least three A3 genes are up regulated by interferon-alpha in human hepatocytes while ectopic expression of activation induced deaminase (AICDA), an A3 paralog, has been noted in a variety of chronic inflammatory syndromes including hepatitis C virus infection. Yet virtually all studies of HBV editing have confined themselves to analyses of virions from culture supernatants or serum where the frequency of edited genomes is generally low (< or = 10(-2)). We decided to look at the nature and frequency of HBV editing in cirrhotic samples taken during removal of a primary hepatocellular carcinoma. Forty-one cirrhotic tissue samples (10 alcoholic, 10 HBV(+), 11 HBV(+)HCV(+) and 10 HCV(+)) as well as 4 normal livers were studied. Compared to normal liver, 5/7 APOBEC3 genes were significantly up regulated in the order: HCV+/-HBV>HBV>alcoholic cirrhosis. A3C and A3D were up regulated for all groups while the interferon inducible A3G was over expressed in virus associated cirrhosis, as was AICDA in approximately 50% of these HBV/HCV samples. While AICDA can indeed edit HBV DNA ex vivo, A3G is the dominant deaminase in vivo with up to 35% of HBV genomes being edited. Despite these highly deleterious mutant spectra, a small fraction of genomes survive and contribute to loss of HBeAg antigenemia and possibly HBsAg immune escape. In conclusion, the cytokine storm associated with chronic inflammatory responses to HBV and HCV clearly up regulates a number of A3 genes with A3G clearly being a major restriction factor for HBV. Although the mutant spectrum resulting from A3 editing is highly deleterious, a very small part, notably the lightly edited genomes, might help the virus evolve and even escape immune responses.
Assuntos
Citidina Desaminase/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B/imunologia , Hepatite B/virologia , Desaminase APOBEC-3G , Idoso , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/genética , Evolução Molecular , Feminino , Regulação Viral da Expressão Gênica/imunologia , Genoma Viral , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Humanos , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Regulação para Cima/fisiologiaRESUMO
Viruses have evolved a plethora of mechanisms to impair host innate immune responses. Herpes simplex virus type 1 (HSV-1), a double-stranded linear DNA virus, impairs the mitochondrial network and dynamics predominantly through the UL12.5 gene. We demonstrated that HSV-1 infection induced a remodeling of mitochondrial shape, resulting in a fragmentation of the mitochondria associated with a decrease in their volume and an increase in their sphericity. This damage leads to the release of mitochondrial DNA (mtDNA) to the cytosol. By generating a stable THP-1 cell line expressing the DNase I-mCherry fusion protein and a THP-1 cell line specifically depleted of mtDNA upon ethidium bromide treatment, we showed that cytosolic mtDNA contributes to type I interferon and APOBEC3A upregulation. This was confirmed by using an HSV-1 strain (KOS37 UL98-SPA) with a deletion of the UL12.5 gene that impaired its ability to induce mtDNA stress. Furthermore, by using an inhibitor of RNA polymerase III, we demonstrated that upon HSV-1 infection, cytosolic mtDNA enhanced type I interferon induction through the RNA polymerase III/RIG-I pathway. APOBEC3A was in turn induced by interferon. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5'TpCpG context. These data demonstrate that upon HSV-1 infection, the mitochondrial network is disrupted, leading to the release of mtDNA and ultimately to its catabolism through APOBEC3-induced mutations. IMPORTANCE Herpes simplex virus 1 (HSV-1) impairs the mitochondrial network through the viral protein UL12.5. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA) in a mouse model. We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of proinflammatory cytokines. The expression of the human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded DNA substrates, resulting in the catabolism of edited DNA. Using human cell lines deprived of mtDNA and viral strains deficient in UL12, we demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that HSV-1 induces mitochondrial network fragmentation in a human model and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.
Assuntos
Proteína DEAD-box 58/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Interferon beta/metabolismo , Mitocôndrias/virologia , RNA Polimerase III/metabolismo , Receptores Imunológicos/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Proteína DEAD-box 58/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno , Humanos , Interferon beta/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Polimerase III/genética , Receptores Imunológicos/genética , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
APOBEC3 (A3) enzymes are best known for their role as antiviral restriction factors and as mutagens in cancer. Although four of them, A3A, A3B, A3F and A3G, are induced by type-1-interferon (IFN-I), their role in inflammatory conditions is unknown. We thus investigated the expression of A3, and particularly A3A and A3B because of their ability to edit cellular DNA, in Systemic Lupus Erythematosus (SLE), a chronic inflammatory disease characterized by high IFN-α serum levels. In a cohort of 57 SLE patients, A3A and A3B, but also A3C and A3G, were upregulated ~ 10 to 15-fold (> 1000-fold for A3B) compared to healthy controls, particularly in patients with flares and elevated serum IFN-α levels. Hydroxychloroquine, corticosteroids and immunosuppressive treatment did not reverse A3 levels. The A3AΔ3B polymorphism, which potentiates A3A, was detected in 14.9% of patients and in 10% of controls, and was associated with higher A3A mRNA expression. A3A and A3B mRNA levels, but not A3C or A3G, were correlated positively with dsDNA breaks and negatively with lymphopenia. Exposure of SLE PBMCs to IFN-α in culture induced massive and sustained A3A levels by 4 h and led to massive cell death. Furthermore, the rs2853669 A > G polymorphism in the telomerase reverse transcriptase (TERT) promoter, which disrupts an Ets-TCF-binding site and influences certain cancers, was highly prevalent in SLE patients, possibly contributing to lymphopenia. Taken together, these findings suggest that high baseline A3A and A3B levels may contribute to cell frailty, lymphopenia and to the generation of neoantigens in SLE patients. Targeting A3 expression could be a strategy to reverse cell death and the generation of neoantigens.