RESUMO
Defective interfering particles (DIPs) are particles containing defective viral genomes (DVGs) generated during viral replication. DIPs have been found in various RNA viruses, especially in influenza viruses. Evidence indicates that DIPs interfere with the replication and encapsulation of wild-type viruses, namely standard viruses (STVs) that contain full-length viral genomes. DIPs may also activate the innate immune response by stimulating interferon synthesis. In this review, the underlying generation mechanisms and characteristics of influenza virus DIPs are summarized. We also discuss the potential impact of DIPs on the immunogenicity of live attenuated influenza vaccines (LAIVs) and development of influenza vaccines based on NS1 gene-defective DIPs. Finally, we review the antiviral strategies based on influenza virus DIPs that have been used against both influenza virus and SARS-CoV-2. This review provides systematic insights into the theory and application of influenza virus DIPs.
Assuntos
COVID-19 , Vacinas contra Influenza , Orthomyxoviridae , Humanos , Antivirais , Vírus Defeituosos Interferentes , Vírus Defeituosos/fisiologia , SARS-CoV-2 , Orthomyxoviridae/genética , Replicação Viral/genéticaRESUMO
Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.
Assuntos
Vírus Defeituosos/fisiologia , Vírion/fisiologia , Animais , Ensaio de Unidades Formadoras de Colônias , Genoma Viral , Humanos , Microscopia Eletrônica de Transmissão , Ensaio de Placa Viral , Viroses/virologia , Replicação ViralRESUMO
During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.
Assuntos
Coinfecção/virologia , Vírus Defeituosos , Modelos Teóricos , Poliovirus , Replicação Viral/fisiologia , Vírus Defeituosos/fisiologia , Humanos , Poliovirus/fisiologiaRESUMO
Respiratory syncytial virus (RSV) causes respiratory illness in children, immunosuppressed individuals and the elderly. However, the viral factors influencing the clinical outcome of RSV infections remain poorly defined. Defective viral genomes (DVGs) can suppress virus replication by competing for viral proteins and by stimulating antiviral immunity. We studied the association between detection of DVGs of the copy-back type and disease severity in three RSV A-confirmed cohorts. In hospitalized children, detection of DVGs in respiratory samples at or around the time of admission associated strongly with more severe disease, higher viral load and a stronger pro-inflammatory response. Interestingly, in experimentally infected adults, the presence of DVGs in respiratory secretions differentially associated with RSV disease severity depending on when DVGs were detected. Detection of DVGs early after infection associated with low viral loads and mild disease, whereas detection of DVGs late after infection, especially if DVGs were present for prolonged periods, associated with high viral loads and severe disease. Taken together, we demonstrate that the kinetics of DVG accumulation and duration could predict clinical outcome of RSV A infection in humans, and thus could be used as a prognostic tool to identify patients at risk of worse clinical disease.
Assuntos
Vírus Defeituosos/genética , Genoma Viral , Mucosa Nasal/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Estudos de Coortes , Vírus Defeituosos/fisiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mucosa Nasal/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/fisiologiaAssuntos
Vírus Defeituosos/genética , Vírus Defeituosos/metabolismo , Orthomyxoviridae/metabolismo , Animais , Vírus Defeituosos/fisiologia , Humanos , Influenza Humana/genética , Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/genética , RNA Viral/genética , RNA Viral/metabolismo , Vírion/genética , Vírion/metabolismo , Replicação Viral/genéticaRESUMO
The host-vector shuttle and the bottleneck in dengue transmission is a significant aspect with regard to the study of dengue outbreaks. As mosquitoes require 100-1000 times more virus to become infected than human, the transmission of dengue virus from human to mosquito is a vulnerability that can be targeted to improve disease control. In order to capture the heterogeneity in the infectiousness of an infected patient population towards the mosquito population, we calibrate a population of host-to-vector virus transmission models based on an experimentally quantified infected fraction of a mosquito population. Once the population of models is well-calibrated, we deploy a population of controls that helps to inhibit the human-to-mosquito transmission of the dengue virus indirectly by reducing the viral load in the patient body fluid. We use an optimal bang-bang control on the administration of the defective virus (transmissible interfering particles (TIPs)) to symptomatic patients in the course of their febrile period and observe the dynamics in successful reduction of dengue spread into mosquitoes.
Assuntos
Vírus Defeituosos/fisiologia , Vírus da Dengue/fisiologia , Dengue/prevenção & controle , Dengue/transmissão , Mosquitos Vetores/virologia , Aedes/virologia , Animais , Dengue/virologia , Humanos , Modelos Teóricos , Carga Viral , Viremia/virologiaRESUMO
Neurotropic viral transsynaptic tracing is an increasingly powerful technique for dissecting the structure and function of neural circuits. Herpes simplex virus type 1 strain H129 has been widely used as an anterograde tracer. However, HSV tracers still have several shortcomings, including high toxicity, low sensitivity and non-specific retrograde labeling. Here, we aimed to construct high-brightness HSV anterograde tracers by increasing the expression of exogenous genes carried by H129 viruses. Using a Trojan horse-like strategy, a HSV/AAV (adeno-associated virus) chimaera termed H8 was generated to enhance the expression of a fluorescent marker. In vitro and in vivo assays showed that the exogenous gene was efficiently replicated and amplified by the synergism of the HSV vector and introduced AAV replication system. H8 reporting fluorescence was brighter than that of currently available H129 tracers, and H8 could be used for fast and effective anterograde tracing without additional immunostaining. These results indicated that foreign gene expression in HSV tracers could be enhanced by integrating HSV with AAV replication system. This approach may be useful as a general enhanced expression strategy for HSV-based tracing tools or gene delivery vectors.
Assuntos
Transporte Axonal/fisiologia , Encéfalo/citologia , Vírus Defeituosos/fisiologia , Dependovirus/fisiologia , Proteínas de Fluorescência Verde/análise , Vírus Auxiliares/fisiologia , Herpesvirus Humano 1/fisiologia , Vias Neurais/ultraestrutura , Técnicas de Rastreamento Neuroanatômico/métodos , Marcadores do Trato Nervoso/análise , Neurônios/ultraestrutura , Vírus Reordenados/fisiologia , Animais , Linhagem Celular , Núcleo Celular/virologia , Vírus Defeituosos/genética , Dependovirus/genética , Genes Reporter , Genes Sintéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Vírus Auxiliares/genética , Herpesvirus Humano 1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/virologia , Vírus Reordenados/genética , Proteínas do Complexo da Replicase Viral/genética , Replicação ViralRESUMO
Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.
Assuntos
Poliaminas Biogênicas/imunologia , Infecções por Bunyaviridae/imunologia , Vírus Defeituosos/fisiologia , Vírus da Encefalite da Califórnia/fisiologia , Vírus da Febre do Vale do Rift/fisiologia , Vírion/fisiologia , Replicação Viral/imunologia , Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/patologia , Linhagem Celular Tumoral , HumanosRESUMO
Defective viral genomes (DVGs) are generated during viral replication and are unable to carry out a full replication cycle unless coinfected with a full-length virus. DVGs are produced by many viruses, and their presence correlates with alterations in infection outcomes. Historically, DVGs were studied for their ability to interfere with standard virus replication as well as for their association with viral persistence. More recently, a critical role for DVGs in inducing the innate immune response during infection was appreciated. Here we review the role of DVGs of RNA viruses in shaping outcomes of experimental as well as natural infections and explore the mechanisms by which DVGs impact infection outcome.
Assuntos
Vírus Defeituosos/genética , Genoma Viral , Vírus de RNA/genética , Viroses/imunologia , Animais , Vírus Defeituosos/fisiologia , Humanos , Imunidade Inata , Vírus de RNA/fisiologia , Viroses/virologia , Replicação ViralRESUMO
Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus-based antivirals.
Assuntos
Vírus Defeituosos/genética , Vírus da Influenza A/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Replicação Viral/genética , Animais , Antivirais/uso terapêutico , Vírus Defeituosos/fisiologia , Cães , Células HEK293 , Humanos , Influenza Humana/tratamento farmacológico , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/tratamento farmacológicoRESUMO
A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Portador Sadio/virologia , Vírus Defeituosos/isolamento & purificação , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Provírus/isolamento & purificação , Latência Viral , Linfócitos T CD4-Positivos/citologia , Portador Sadio/terapia , Linhagem Celular , DNA Viral/análise , DNA Viral/genética , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Infecções por HIV/terapia , HIV-1/genética , HIV-1/fisiologia , Humanos , Ativação Linfocitária , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/fisiologiaRESUMO
Retroviral pseudotypes are broadly used as safe instruments to mimic the structure and surface of highly pathogenic viruses. They have been employed for the discovery of new drugs, as diagnostic tools in vaccine studies, and part of serological assays. Because of their widespread use in research and their potential as tools for quality control, it is important to know their shelf life, stability, and best storage conditions. In this study, we produced pseudotypes carrying the lacZ reporter gene and the hemagglutinin (HA) of avian influenza virus subtypes H5 and H7 to investigate their stability under various storage conditions. We produced pseudotypes with titers of approximately 106 RLU/mL, which decreased to 105-106 RLU/mL after short-term storage at 4 °C (up to 4 weeks). Stability was maintained after long-term storage at -20 °C (up to 12 months), even under storage variations such as freeze-thaw cycles. We conclude that, although the titers decreased by 1 log10 under the different storage conditions, the remaining titers can be readily applicable in many techniques, such as neutralization assays. These findings show that large quantities of retroviral pseudotypes can be safely stored for short- or long-term use, allowing standardization and reduced variation in assays involving retroviral pseudotypes.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Lentivirus/fisiologia , Temperatura Baixa , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Genes Reporter/genética , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Células HEK293 , Humanos , Lentivirus/genéticaRESUMO
Influenza B virus (FLUBV) is an important pathogen that infects humans and causes seasonal influenza epidemics. To date, little is known about defective genomes of FLUBV and their roles in viral replication. In this study, by using a next-generation sequencing approach, we analyzed total mRNAs extracted from A549 cells infected with B/Brisbane/60/2008 virus (Victoria lineage), and identified four defective FLUBV genomes with two (PB1∆A and PB1∆B) from the polymerase basic subunit 1 (PB1) segment and the other two (M∆A and M∆B) from the matrix (M) protein-encoding segment. These defective genomes contained significant deletions in the central regions with each having the potential for encoding a novel polypeptide. Significantly, each of the discovered defective RNAs can potently inhibit the replication of B/Yamanashi/166/98 (Yamagata lineage). Furthermore, PB1∆A was able to interfere modestly with influenza A virus (FLUAV) replication. In summary, our study provides important initial insights into FLUBV defective-interfering genomes, which can be further explored to achieve better understanding of the replication, pathogenesis and evolution of FLUBV.
Assuntos
Vírus Defeituosos/genética , Vírus da Influenza B/genética , Influenza Humana/virologia , RNA Viral/genética , Células A549 , Vírus Defeituosos/fisiologia , Genoma Viral , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Proteínas Virais/genética , Replicação ViralRESUMO
A defective interfering particle (DIP) in the context of influenza A virus is a virion with a significantly shortened RNA segment substituting one of eight full-length parent RNA segments, such that it is preferentially amplified. Hence, a cell co-infected with DIPs will produce mainly DIPs, suppressing infectious virus yields and affecting infection kinetics. Unfortunately, the quantification of DIPs contained in a sample is difficult because they are indistinguishable from standard virus (STV). Using a mathematical model, we investigated the standard experimental method for counting DIPs based on the reduction in STV yield (Bellett & Cooper, 1959, Journal of General Microbiology 21, 498-509 (doi:10.1099/00221287-21-3-498)). We found the method is valid for counting DIPs provided that: (i) an STV-infected cell's co-infection window is approximately half its eclipse phase (it blocks infection by other virions before it begins producing progeny virions), (ii) a cell co-infected by STV and DIP produces less than 1 STV per 1000 DIPs and (iii) a high MOI of STV stock (more than 4 PFU per cell) is added to perform the assay. Prior work makes no mention of these criteria such that the method has been applied incorrectly in several publications discussed herein. We determined influenza A virus meets these criteria, making the method suitable for counting influenza A DIPs.
Assuntos
Vírus Defeituosos/fisiologia , Vírus da Influenza A/fisiologia , Modelos Biológicos , Vírion/fisiologia , HumanosRESUMO
UNLABELLED: African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis. IMPORTANCE: African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated. These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. Importantly, these candidate vaccine strains showed strong protective efficacy against AHSV infection in an IFNAR(-/-) mouse model.
Assuntos
Vírus da Doença Equina Africana/imunologia , Doença Equina Africana/prevenção & controle , Vírus Defeituosos/imunologia , Vacinas Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus , Replicação Viral , Vírus da Doença Equina Africana/genética , Vírus da Doença Equina Africana/fisiologia , Animais , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Modelos Animais de Doenças , Deleção de Genes , Camundongos , Camundongos Knockout , Genética Reversa , Sorogrupo , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
UNLABELLED: When viruses infect their host cells, they can make defective virus-like particles along with intact virus. Cells coinfected with virus and defective particles often exhibit interference with virus growth caused by the competition for resources by defective genomes. Recent reports of the coexistence and cotransmission of such defective interfering particles (DIPs) in vivo, across epidemiological length and time scales, suggest a role in viral pathogenesis, but it is not known how DIPs impact infection spread, even under controlled culture conditions. Using fluorescence microscopy, we quantified coinfections of vesicular stomatitis virus (VSV) expressing a fluorescent reporter protein and its DIPs on BHK-21 host cell monolayers. We found that viral gene expression was more delayed, infections spread more slowly, and patterns of spread became more "patchy" with higher DIP inputs to the initial cell. To examine how infection spread might depend on the behavior of the initial coinfected cell, we built a computational model, adapting a cellular automaton (CA) approach to incorporate kinetic data on virus growth for the first time. Specifically, changes in observed patterns of infection spread could be directly linked to previous high-throughput single-cell measures of virus-DIP coinfection. The CA model also provided testable hypotheses on the spatial-temporal distribution of the DIPs, which remain governed by their predator-prey interaction. More generally, this work offers a data-driven computational modeling approach for better understanding of how single infected cells impact the multiround spread of virus infections across cell populations. IMPORTANCE: Defective interfering particles (DIPs) compete with intact virus, depleting host cell resources that are essential for virus growth and infection spread. However, it is not known how such competition, strong or weak, ultimately affects the way in which infections spread and cause disease. In this study, we address this unmet need by developing an integrated experimental-computational approach, which sheds new light on how infections spread. We anticipate that our approach will also be useful in the development of DIPs as therapeutic agents to manage the spread of viral infections.
Assuntos
Vírus Defeituosos/fisiologia , Vesiculovirus/fisiologia , Replicação Viral , Animais , Linhagem Celular , Cricetinae , Células Epiteliais/virologia , Microscopia de Fluorescência , Análise Espaço-Temporal , Vesiculovirus/crescimento & desenvolvimento , Interferência ViralRESUMO
We have generated defective Viral Hemorrhagic Septicemia Viruses (VHSV) which express either the green fluorescent protein (GFP) or a far-red fluorescent protein (mKate) by replacing the genes encoding the nucleoprotein N or the polymerase-associated P protein. To recover viable defective viruses, rVHSV-ΔN-Red and rVHSV-ΔP-Green, fish cells were co-transfected with both deleted cDNA VHSV genomes, together with plasmids expressing N, P and L of the RNA-dependent RNA polymerase. After one passage of the transfected cell supernatant, red and green cell foci were observed. Viral titer reached 107 PFU/mL after three passages. Infected cells were always red and green with the very rare event of single red or green cell foci appearing. To clarify our understanding of how such defective viruses could be so efficiently propagated, we investigated whether (i) a recombination event between both defective genomes had occurred, (ii) whether both genomes were co-encapsidated in a single viral particle, and (iii) whether both defective viruses were always replicated together through a complementation phenomenon or even as conglomerate. To address these hypotheses, genome and viral particles have been fully characterized and, thus, allowing us to conclude that rVHSV-ΔN-Red and rVHSV-ΔP-Green are independent viral particles which could propagate only by simultaneously infecting the same cells.
Assuntos
Vírus Defeituosos/fisiologia , Novirhabdovirus/fisiologia , Replicação Viral , Animais , Células Cultivadas , Vírus Defeituosos/genética , Peixes , Deleção de Genes , Genes Reporter/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Novirhabdovirus/genética , Plasmídeos , Inoculações Seriadas , Coloração e Rotulagem , Carga Viral , Ensaio de Placa Viral , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína Vermelha FluorescenteRESUMO
Melanoma differentiation-associated gene 5 (MDA-5, IFIH1), a cytosolic innate pattern recognition receptor, functions as a first line of defense against viral infection by sensing double-stranded RNA. Ectopic expression of MDA-5 has been shown to induce cancer cell death, but the mechanism of action by which MDA-5 exerts these cytotoxic effects is unclear. Here, we demonstrate that ectopic expression of MDA-5 via replication-incompetent adenovirus (Ad.Mda-5) initiates multiple signaling cascades, culminating in cytotoxicity and type I IFN production in mouse and human prostate cancer cells. This intrinsic dual activity of MDA-5 required the adaptor protein IFNß promoter stimulator 1 (IPS-1, MAVS) and could be functionally uncoupled. MDA-5 lacking N-terminal caspase recruitment domains (CARD) engaged an intracellular death program in cancer cells but was unable to efficiently stimulate the expression of IFNß. In contrast to cancer cells susceptible to MDA-5-mediated cytotoxicity, normal cells were highly resistant and instead developed a robust type I IFN response. Strikingly, intratumoral delivery of Ad.Mda-5 led to regression of preestablished prostate cancers and development of long-lasting antitumor immune memory, which was primarily attributed to the activation of tumor-reactive cytotoxic T lymphocytes and/or natural killer cells. Using the CARD-truncated MDA-5 mutant, silencing of IPS-1, and antibody blockade of the IFNα/ß receptor, we further demonstrate that type I IFN signaling was crucial for in situ MDA-5-induced protective antitumor immunity. Therefore, deliberately targeting the evolutionarily conserved MDA-5-IPS-1 antiviral pathway in tumors can provoke parallel tumoricidal and immunostimulatory effects that bridge innate and adaptive immune responses for the therapeutic treatment of cancer. Cancer Res; 76(8); 2166-76. ©2016 AACR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adenoviridae/fisiologia , Apoptose/fisiologia , Vírus Defeituosos/fisiologia , Interferon Tipo I/fisiologia , Helicase IFIH1 Induzida por Interferon/fisiologia , Neoplasias da Próstata/patologia , Animais , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/imunologiaRESUMO
Primary pain and touch sensory neurons not only detect internal and external sensory stimuli, but also receive inputs from other neurons. However, the neuronal derived inputs for primary neurons have not been systematically identified. Using a monosynaptic rabies viruses-based transneuronal tracing method combined with sensory-specific Cre-drivers, we found that sensory neurons receive intraganglion, intraspinal, and supraspinal inputs, the latter of which are mainly derived from the rostroventral medulla (RVM). The viral-traced central neurons were largely inhibitory but also consisted of some glutamatergic neurons in the spinal cord and serotonergic neurons in the RVM. The majority of RVM-derived descending inputs were dual GABAergic and enkephalinergic (opioidergic). These inputs projected through the dorsolateral funiculus and primarily innervated layers I, II, and V of the dorsal horn, where pain-sensory afferents terminate. Silencing or activation of the dual GABA/enkephalinergic RVM neurons in adult animals substantially increased or decreased behavioral sensitivity, respectively, to heat and mechanical stimuli. These results are consistent with the fact that both GABA and enkephalin can exert presynaptic inhibition of the sensory afferents. Taken together, this work provides a systematic view of and a set of tools for examining peri- and extrasynaptic regulations of pain-afferent transmission.
Assuntos
Vias Aferentes/fisiologia , Vias Eferentes/fisiologia , Rede Nervosa/fisiologia , Nociceptividade/fisiologia , Células Receptoras Sensoriais/fisiologia , Corno Dorsal da Medula Espinal/citologia , Animais , Vírus Defeituosos/fisiologia , Encefalinas/fisiologia , Membro Anterior/inervação , Neurônios GABAérgicos/fisiologia , Neurônios GABAérgicos/virologia , Gânglios Espinais/citologia , Hiperalgesia/fisiopatologia , Interneurônios/fisiologia , Interneurônios/virologia , Proteínas do Tecido Nervoso/análise , Condução Nervosa , Neurônios Aferentes/fisiologia , Neurônios Aferentes/virologia , Neurônios Eferentes/fisiologia , Neurônios Eferentes/virologia , Nociceptores/fisiologia , Células do Corno Posterior/fisiologia , Células do Corno Posterior/virologia , Terminações Pré-Sinápticas/fisiologia , Vírus da Raiva/fisiologia , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/virologia , Pele/inervação , Corno Dorsal da Medula Espinal/fisiologia , Corno Dorsal da Medula Espinal/ultraestrutura , Replicação Viral , Ácido gama-Aminobutírico/fisiologiaRESUMO
We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.