RESUMO
The FDA has recently approved Krystal biotech's beremagene geperpavec (B-VEC, Vyjuvek) to treat the wounds of dystrophic epidermolysis bullosa (DEB) patients. This represents a giant step, not only toward the treatment of this devastating disease, but also for the whole field of non-replicative (nr) recombinant HSV-1 vectors for gene therapy. To view this Bench to Bedside, open or download the PDF.
Assuntos
Epidermólise Bolhosa Distrófica , Terapia Genética , Humanos , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Herpesvirus Humano 1/genéticaRESUMO
The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.
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Doença de Alzheimer , Anticorpos de Cadeia Única , Camundongos , Ratos , Humanos , Animais , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Doença de Alzheimer/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Sinapses/metabolismo , Neurônios/metabolismo , Transtornos da Memória/genética , Transtornos da Memória/terapiaRESUMO
BACKGROUND: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. METHODS: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters' candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. RESULTS: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. CONCLUSIONS: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.
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Herpes Simples , Herpesvirus Humano 1 , Traumatismos da Medula Espinal , Bexiga Urinária Hiperativa , Animais , Gânglios Espinais/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Ratos , Células Receptoras Sensoriais/metabolismo , Traumatismos da Medula Espinal/metabolismo , Bexiga Urinária Hiperativa/terapiaRESUMO
Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure, allowing its interaction with many ligands, including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+ T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect, which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defense mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of HEp-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus 2 (HSV-2), respectively, suggesting that HRG might display broad antiviral activity under acidic conditions.IMPORTANCE Vaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated to be 1 in every 1,000 episodes of sexual intercourse, reflecting the high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses, such as respiratory syncytial virus and herpes simplex virus 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/prevenção & controle , Proteínas/farmacologia , Animais , Antivirais , Proteínas Sanguíneas , Linhagem Celular , Muco do Colo Uterino/química , Muco do Colo Uterino/metabolismo , Chlorocebus aethiops , Feminino , Glicoproteínas/metabolismo , Glicoproteínas/farmacologia , HIV-1/metabolismo , Heparitina Sulfato/metabolismo , Herpesvirus Humano 2/metabolismo , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Proteínas/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Células Vero , Viroses/metabolismo , Viroses/prevenção & controleRESUMO
Nucleolin (NCL) is a major component of the cell nucleolus, which has the ability to rapidly shuttle to several other cells' compartments. NCL plays important roles in a variety of essential functions, among which are ribosome biogenesis, gene expression, and cell growth. However, the precise mechanisms underlying NCL functions are still unclear. Our study aimed to provide new information on NCL functions via the identification of its nuclear interacting partners. Using an interactomics approach, we identified 140 proteins co-purified with NCL, among which 100 of them were specifically found to be associated with NCL after RNase digestion. The functional classification of these proteins confirmed the prominent role of NCL in ribosome biogenesis and additionally revealed the possible involvement of nuclear NCL in several pre-mRNA processing pathways through its interaction with RNA helicases and proteins participating in pre-mRNA splicing, transport, or stability. NCL knockdown experiments revealed that NCL regulates the localization of EXOSC10 and the amount of ZC3HAV1, two components of the RNA exosome, further suggesting its involvement in the control of mRNA stability. Altogether, this study describes the first nuclear interactome of human NCL and provides the basis for further understanding the mechanisms underlying the essential functions of this nucleolar protein.
Assuntos
Fosfoproteínas/fisiologia , Proteômica/métodos , Proteínas de Ligação a RNA/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/química , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Ribossomos , NucleolinaRESUMO
Following primary infections HSV-1 replicates productively in epithelial cells and enters sensory neurons via nerve termini. After retrograde transport the virus genome is delivered into the cell nucleus, where it establishes lifelong latent infections. During latency, the virus genome remains as a chromatinized episome expressing only a set of latency-associated transcripts (LAT) and a group of microRNAs that inhibit expression of key lytic viral functions. Periodically the virus can reactivate to reinitiate lytic, secondary infections at peripheral tissues. The ability to establish both lytic and latent infections relies on the coexistence in the virus genome of two alternative gene expression programs, under the control of epigenetic mechanisms. Latency is an adaptive phenotype that allows the virus to escape immune host responses and to reactivate and disseminate to other hosts upon recognizing danger signals such as stress, neurologic trauma or growth factor deprivation.
Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Ativação Viral/fisiologia , Latência Viral/fisiologia , Capsídeo/fisiologia , Efeito Citopatogênico Viral/fisiologia , Epigênese Genética , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica , Genes Virais , Herpes Simples/imunologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/genética , MicroRNAs/fisiologia , RNA Viral/fisiologia , Células Receptoras Sensoriais/virologia , Estresse Fisiológico , Proteínas Virais/genética , Proteínas Virais/fisiologia , Internalização do Vírus , Liberação de Vírus/fisiologia , Replicação Viral/fisiologiaRESUMO
Herpes simplex virus type 1 (HSV-1) is a DNA virus and human pathogen used to construct promising therapeutic vectors. HSV-1 vectors fall into two classes: replication-selective oncolytic vectors for cancer therapy and defective non-replicative vectors for gene therapy. Vectors from each class can accommodate ≥30 kb of inserts, have been approved clinically, and demonstrate a relatively benign safety profile. Despite oncolytic HSV (oHSV) replication in tumors and elicited immune responses, the virus is well tolerated in cancer patients. Current non-replicative vectors elicit only limited immune responses. Seropositivity and immune responses against HSV-1 do not eliminate either the vector or infected cells, and the vectors can therefore be re-administered. In this review we highlight vectors that have been translated to the clinic and host-virus immune interactions that impact on the safety and efficacy of HSVs.
Assuntos
Vetores Genéticos , Herpesvirus Humano 1 , Terapia Viral Oncolítica , Vírus Oncolíticos , Replicação Viral , Humanos , Vetores Genéticos/genética , Terapia Viral Oncolítica/métodos , Animais , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Neoplasias/terapia , Neoplasias/imunologia , Terapia Genética/métodos , Terapia Genética/efeitos adversosRESUMO
Herpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection.
Assuntos
Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Far-Western Blotting , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno , NucleolinaRESUMO
Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Herpesvirus Humano 1/imunologia , Infecções por Rotavirus/prevenção & controle , Rotavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Chlorocebus aethiops , Feminino , Vetores Genéticos , Células HEK293 , Herpesvirus Humano 1/genética , Humanos , Imunidade Humoral , Imunidade nas Mucosas , Imunização , Camundongos , Rotavirus/genética , Infecções por Rotavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Células Vero , Vírion/genética , Vírion/imunologiaRESUMO
Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Proteínas de Ligação a RNA/metabolismo , Algoritmos , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/virologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Nectinas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Interferência de RNA , Proteínas de Ligação a RNA/genética , Estatísticas não Paramétricas , Espectrometria de Massas em Tandem , Eletroforese em Gel Diferencial Bidimensional , Ensaio de Placa Viral , Proteínas Virais/metabolismoRESUMO
Among the variety of viral vectors, those derived from the human parvovirus Adeno-Associated Virus (AAV) have emerged as a very efficient tool for in vivo gene transfer into a variety of tissues and animal species during the two last decades. The relative simplicity of the organization of the AAV genome and the non-pathogenic property of the parental AAV has greatly contributed to the use of this viral vector among the gene transfer community. However, the limited knowledge of the wild type (wt) virus compared to other viral vectors has required considerable efforts to gain insight into wt AAV biology in order to improve the AAV vector system for therapy. This review will summarize the most important features of both wt and recombinant AAV to show how the increased understanding of the biology of the virus has enabled AAV vectors to lead the in vivo gene transfer field.
RESUMO
Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Dependovirus/fisiologia , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Infecções por Parvoviridae/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Replicação do DNA , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Células HeLa , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Infecções por Parvoviridae/virologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Células Vero , Proteínas Virais/genéticaRESUMO
The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.
Assuntos
Replicação do DNA/fisiologia , Dependovirus/fisiologia , Regulação Viral da Expressão Gênica/genética , Vírus Auxiliares/fisiologia , Herpesvirus Humano 1/fisiologia , Replicação Viral/fisiologia , Animais , Southern Blotting , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Imunofluorescência , Células HeLa , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Hibridização in Situ Fluorescente , Plasmídeos/genética , Reação em Cadeia da Polimerase , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Diverse oncolytic viruses (OV) are being designed for the treatment of cancer. The characteristics of the parental virus strains may influence the properties of these agents. AIMS: To characterize two herpes simplex virus 1 strains (HSV-1 17syn(+) and HFEM) as platforms for virotherapy against liver cancer. METHODS: The luciferase reporter gene was introduced in the intergenic region 20 locus of both HSV-1 strains, giving rise to the Cgal-Luc and H6-Luc viruses. Their properties were studied in hepatocellular carcinoma (HCC) cells in vitro. Biodistribution was monitored by bioluminescence imaging (BLI) in athymic mice and immune-competent Balb/c mice. Immunogenicity was studied by MHC-tetramer staining, in vivo killing assays and determination of specific antibody production. Intratumoural transgene expression and oncolytic effect were studied in HuH-7 xenografts. RESULTS: The H6-Luc virus displayed a syncytial phenotype and showed higher cytolytic effect on some HCC cells. Upon intravenous or intrahepatic injection in mice, both viruses showed a transient transduction of the liver with rapid relocalization of bioluminescence in adrenal glands, spinal cord, uterus and ovaries. No significant differences were observed in the immunogenicity of these viruses. Local intratumoural administration caused progressive increase in transgene expression during the first 5 days and persisted for at least 2 weeks. H6-Luc achieved faster amplification of transgene expression and stronger inhibition of tumour growth than Cgal-Luc, although toxicity of these non-attenuated viruses should be reduced to obtain a therapeutic effect. CONCLUSIONS: The syncytial H6-Luc virus has a strong oncolytic potential on human HCC xenografts and could be the basis for potent OV.
Assuntos
Carcinoma Hepatocelular/terapia , Herpesvirus Humano 1/genética , Neoplasias Hepáticas/terapia , Terapia Viral Oncolítica/métodos , Animais , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Sobrevivência Celular , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Herpesvirus Humano 1/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Hepáticas/virologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase , Estatísticas não Paramétricas , Transdução Genética , Transgenes/genéticaRESUMO
In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.
Assuntos
Carcinoma Hepatocelular/virologia , Herpesvirus Humano 1/fisiologia , Neoplasias Hepáticas/virologia , Organelas/metabolismo , Proteômica , Transdução de Sinais , Replicação Viral , Animais , Apoptose , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Chlorocebus aethiops , Efeito Citopatogênico Viral , Citosol/metabolismo , Citosol/virologia , Ativação Enzimática , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Microssomos/metabolismo , Microssomos/virologia , Modelos Biológicos , Organelas/virologia , Proteína Fosfatase 2/metabolismo , Proteoma/análise , Proteoma/química , Células VeroRESUMO
N-methyl-D-aspartate receptors are heterotetramers composed of two GluN1 obligatory subunits and two regulatory subunits. In cognitive-related brain structures, GluN2A and GluN2B are the most abundant regulatory subunits, and their expression is subjected to tight regulation. During development, GluN2B expression is characteristic of immature synapses, whereas GluN2A is present in mature ones. This change in expression induces a shift in GluN2A/GluN2B ratio known as developmental switch. Moreover, modifications in this relationship have been associated with learning and memory, as well as different pathologies. In this work, we used a specific shRNA to induce a reduction in GluN2A expression after the developmental switch, both in vitro in primary cultured hippocampal neurons and in vivo in adult male Wistar rats. After in vitro characterization, we performed a cognitive profile and evaluated seizure susceptibility in vivo. Our in vitro results showed that the decrease in the expression of GluN2A changes GluN2A/GluN2B ratio without altering the expression of other regulatory subunits. Moreover, rats expressing the anti-GluN2A shRNA in vivo displayed an impaired contextual fear-conditioning memory. In addition, these animals showed increased seizure susceptibility, in terms of both time and intensity, which led us to conclude that deregulation in GluN2A expression at the hippocampus is associated with seizure susceptibility and learning-memory mechanisms.
RESUMO
Soluble amyloid-ß peptide (Aß) oligomers, known to accumulate in Alzheimer's disease brains, target excitatory post-synaptic terminals. This is thought to trigger synapse deterioration, a mechanism possibly underlying memory loss in early stage Alzheimer's disease. A major unknown is the identity of the receptor(s) targeted by oligomers at synapses. Because oligomers have been shown to interfere with N-methyl-d-aspartate receptor (NMDAR) function and trafficking, we hypothesized that NMDARs might be required for oligomer binding to synapses. An amplicon vector was used to knock-down NMDARs in mature hippocampal neurons in culture, yielding 90% reduction in dendritic NMDAR expression and blocking neuronal oxidative stress induced by Aß oligomers, a pathological response that has been shown to be mediated by NMDARs. Remarkably, NMDAR knock-down abolished oligomer binding to dendrites, indicating that NMDARs are required for synaptic targeting of oligomers. Nevertheless, oligomers do not appear to bind directly to NMDARs as indicated by the fact that both oligomer-attacked and non-attacked neurons exhibit similar surface levels of NMDARs. Furthermore, pre-treatment of neurons with insulin down-regulates oligomer-binding sites in the absence of a parallel reduction in surface levels of NMDARs. Establishing that NMDARs are key components of the synaptic oligomer binding complex may illuminate the development of novel approaches to prevent synapse failure triggered by Aß oligomers.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Hipocampo/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Ligação Proteica/fisiologia , Ratos , Receptores de N-Metil-D-Aspartato/deficiência , Sinapses/patologiaRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a cancer of poor prognosis, with limited success in patient treatment, which it makes an excellent target for gene therapy and viral oncolysis. Accordingly, herpes virus simplex type-1 (HSV-1) is one of the most promising viral platforms for transferring therapeutic genes and the development of oncolytic vectors that can target, multiply in, and eradicate hepatoma cells via their lytic cycle. Enhanced efficacy and specificity of HSV-1-based vectors towards HCC may be achieved by using HCC-specific gene promoters to drive selective viral gene expression and accomplish conditional replication and/or to control the expression of therapeutic genes. However, careful verification of promoter function in the context of the replication-competent HSV-1 vectors is required. The present study aimed to identify novel HCC-specific promoters that could efficiently direct transgene expression to HCC cells and maintain their activity during active viral replication. METHODS: Publicly available microarray data from human HCC biopsies were analysed in order to detect novel candidate genes induced primarily in HCC compared to normal liver. HCC specificity and promoter activity were evaluated by RT-PCR and chromatin immunoprecipitation. Additionally, transcriptional activity of promoters was further evaluated in the context of HSV-1 genome, using luciferase assays in cultured cells and animal models. RESULTS: Eight HCC-specific genes were characterised in this study: Angiopoietin-like-3, Cytochrome P450, family 2, subfamily C, polypeptide 8, Vitronectin, Alcohol dehydrogenase 6-class V, Apolipoprotein B, Fibrinogen beta chain, Inter-alpha-globulin-inhibitor H3 and Inter-alpha-globulin-inhibitor H1. Specific HCC expression and active gene transcription were confirmed in human liver and non-liver cell lines and further evaluated in primary neoplastic cells from hepatitis C and B virus (HCV- and HBV)-associated HCC patients. High promoter activity and specificity in the presence of HSV-1 infection and from within the viral genome, was validated, both in vitro and in vivo. CONCLUSIONS: We identified and experimentally characterized novel hepatoma-specific promoters, which were valuable for cancer-specific gene therapy, using HSV-1 vectors.
Assuntos
Carcinoma Hepatocelular/terapia , Sistemas de Liberação de Medicamentos/métodos , Genes Neoplásicos , Terapia Genética/métodos , Vetores Genéticos , Herpesvirus Humano 1/genética , Regiões Promotoras Genéticas , Humanos , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Infection of the brain with various types of pathogens, and the resulting inflammatory response, is becoming increasingly important in our understanding of the etiology of Alzheimer's disease (AD). The fact that several genes identified as risk factors are actually involved in the modulation of the immune response, as well as the very diversity of the infectious agents identified as possible actors in the evolution of this disease, argue in favor of the neuro-inflammatory hypothesis, as does the demonstration that the protein Aß, one of the most important markers of AD, is an antimicrobial peptide. Among others, herpes viruses (mainly, but not only, HSV-1), which can establish latent infections in brain neurons, especially in the elder population, punctuated by episodes of reactivation following stress or immunosuppression, appear as very strong candidates to play an etiological role, if only as cofactors, of AD. Recent results show that, in human and rat neurons, infection with HSV-1 increases the formation of Aß along the amyloidogenic pathway, as well as the phosphorylation of Tau proteins, another essential marker of AD. The growing evidence that chronic infections and defense mechanisms, including inflammatory processes, are at the heart of AD, warrants reviewing antiviral drugs such as acyclovir, and possibly vaccination, as potential avenues for AD control.
TITLE: Maladie d'Alzheimer, neuro-inflammation et virus herpétiques - Une piste qui trace son chemin. ABSTRACT: L'infection du cerveau par divers types d'agents pathogènes, et les réponses inflammatoires qui s'en suivent, occupent une place grandissante dans notre compréhension de l'étiologie de la maladie d'Alzheimer (MA). Le fait que, parmi la vingtaine de gènes identifiés comme étant des facteurs à risque, plusieurs soient impliqués dans la modulation de la réponse immunitaire, ainsi que la diversité même des agents infectieux identifiés comme étant des acteurs possibles dans l'évolution de cette maladie, plaident en faveur de l'hypothèse neuro-inflammatoire, tout comme la prise de conscience que la protéine Aß, l'un des marqueurs les plus importants de la MA, peut agir comme un système de défense antimicrobienne, capable de neutraliser des bactéries et des virus. Différent types de pathogènes, incluant des bactéries, des champignons, des protozoaires et des virus, ont été identifiés dans le cerveau malade, souvent près des lésions caractéristiques de la MA. Parmi eux, les virus herpétiques (surtout, mais pas seulement, HSV-1), qui se caractérisent par l'établissement d'infections latentes dans les neurones, ponctuées par des épisodes de réactivation suite à des stress ou des immunodépressions, apparaissent comme des candidats très solides à un rôle étiologique, ne serait-ce qu'en tant que cofacteurs, de la MA. La présence de génomes HSV-1 latents dans le cerveau, et donc le risque de réactivation, augmentent significativement avec l'âge. Des résultats récents montrent que, dans des neurones humains et de rat, l'infection par HSV-1 augmente l'expression de la ß-sécrétase et de la nicastrine, deux enzymes impliquées dans la formation des Aß selon la voie amyloïdogénique, ainsi que de celle de GSK3ß et PKA, deux kinases impliquées dans la phosphorylation des protéines Tau, un autre marqueur essentiel de la MA. Les preuves croissantes obtenues, selon lesquelles les infections chroniques et les mécanismes de défense suscités, y compris les processus inflammatoires, sont au cÅur de la MA, justifient de revoir les médicaments antiviraux tels que l'acyclovir, et peut-être aussi la vaccination, comme des voies potentielles de lutte contre la MA.
Assuntos
Doença de Alzheimer/etiologia , Herpesviridae/fisiologia , Inflamação/complicações , Neurônios/imunologia , Neurônios/virologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Doença de Alzheimer/virologia , Animais , Antivirais/uso terapêutico , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Humanos , Inflamação/patologia , Inflamação/virologia , Neuroimunomodulação/fisiologia , Neurônios/patologia , Ratos , Fatores de Risco , Transdução de Sinais/fisiologiaRESUMO
Amplicon vectors, or amplicons, are defective, helper-dependent, herpes simplex virus type 1 (HSV-1)-based vectors. The main interest of amplicons as gene transfer tools stems from the fact that the genomes of these vectors do not carry protein-encoding viral sequences. Consequently, they are completely safe for the host and nontoxic for the infected cells. Moreover, the complete absence of virus genes provides a genomic space authorizing a very large payload, enough to accommodate foreign DNA sequences up to almost 150-kbp, the size of the HSV-1 genome. This transgene capacity can be used to deliver complete gene loci, including introns and exons, as well as long regulatory sequences conferring tissue-specific expression or stable maintenance of the transgene in proliferating cells. For many years the development of these vectors and their application in gene transfer experiments was hindered by the presence of contaminating toxic helper virus particles in the vector stocks. In recent years, however, two different methodologies have been developed that allow generating amplicon stocks either completely free of helper particles or only faintly contaminated with fully defective helper particles. This chapter describes these two methodologies.