RESUMO
Exosomes and other extracellular vesicles (EVs) play a significant yet poorly understood role in cell-cell communication during homeostasis and various pathological conditions. Conventional in vitro and in vivo approaches for studying exosome/EV function depend on time-consuming and expensive vesicle purification methods to obtain sufficient vesicle populations. Moreover, the existence of various EV subtypes with distinct functional characteristics and submicron size makes their analysis challenging. To help address these challenges, we present here a unique chip-based approach for real-time monitoring of cellular EV exchange between physically separated cell populations. The extracellular matrix (ECM)-mimicking Matrigel is used to physically separate cell populations confined within microchannels, and mimics tissue environments to enable direct study of exosome/EV function. The submicron effective pore size of the Matrigel allows for the selective diffusion of only exosomes and other smaller EVs, in addition to soluble factors, between co-cultured cell populations. Furthermore, the use of PEGDA hydrogel with a very small pore size of 1.2 nm in lieu of Matrigel allows us to block EV migration and, therefore, differentiate EV effects from effects that may be mediated by soluble factors. This versatile platform bridges purely in vitro and in vivo assays by enabling studies of EV-mediated cellular crosstalk under physiologically relevant conditions, enabling future exosome/EV investigations across multiple disciplines through real-time monitoring of vesicle exchange.
Assuntos
Exossomos , Vesículas Extracelulares , Comunicação Celular , Células Cultivadas , MicrofluídicaRESUMO
A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , Quinases Lim/antagonistas & inibidores , Liberação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/isolamento & purificação , Células Cultivadas , Ebolavirus/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/isolamento & purificação , HIV-1/fisiologia , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Vírus da Febre do Vale do Rift/efeitos dos fármacosRESUMO
HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/µl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-ß, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Citocinas/metabolismo , Exossomos/metabolismo , HIV-1/imunologia , Leucócitos/metabolismo , MicroRNAs/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Células Cultivadas , Exossomos/imunologia , Exossomos/virologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Interleucina-6/metabolismo , Leucócitos/imunologia , Leucócitos/virologia , Linfotoxina-alfa/metabolismo , Camundongos Endogâmicos NOD , Camundongos Transgênicos , MicroRNAs/sangue , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possesses a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival. However, the role of S-nitrosylation of host cell proteins during B. anthracis infection has not been determined. METHODS: Nitrosoproteomic analysis of human small airway epithelial cells (HSAECs) infected with toxigenic B. anthracis Sterne was performed, identifying peroxiredoxin 1 (Prx1) as one predominant target. Peroxidase activity of Prx during infection was measured using 2-Cys-Peroxiredoxin activity assay. Chaperone activity of S-nitrosylated Prx1 was measured by insulin aggregation assay, and analysis of formation of multimeric species using Native PAGE. Griess assay and DAF-2DA fluorescence assay were used to measure NO production. Cell viability was measured using the Alamar Blue assay and the ATPlite assay (Perkin Elmer). RESULTS: S-nitrosylation of Prx1 in Sterne-infected HSAECs leads to a decrease in its peroxidase activity while enhancing its chaperone function. Treatment with bNOS inhibitor, or infection with bNOS deletion strain, reduces S-nitrosylation of Prx1 and decreases host cell survival. Consistent with this, siRNA knockdown of Prx1 lowers bNOS-dependent protection of HSAEC viability. CONCLUSIONS: Anthrax infection results in S-nitrosylation of multiple host proteins, including Prx1. The nitrosylation-dependent decrease in peroxidase activity of Prx1 and increase in its chaperone activity is one factor contributing to enhancing infected cell viability. GENERAL SIGNIFICANCE: These results provide a new venue of mechanistic investigation for inhalational anthrax that could lead to novel and potentially effective countermeasures.
Assuntos
Antraz/microbiologia , Antraz/patologia , Bacillus anthracis/patogenicidade , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Pulmão/patologia , Peroxirredoxinas/metabolismo , Bacillus anthracis/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Deleção de Genes , Humanos , Espectrometria de Massas , Modelos Biológicos , Chaperonas Moleculares/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Nitrosação , Peroxidase/metabolismo , Reprodutibilidade dos TestesRESUMO
During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10(fl); Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitf(vga9) ) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitf(vga9) does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage.
Assuntos
Desenvolvimento Embrionário/genética , Melanócitos/citologia , Fatores de Transcrição SOXE/genética , Células-Tronco/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Cor de Cabelo/genética , Melanócitos/metabolismo , Camundongos , Camundongos Knockout , Fatores de Transcrição SOXE/metabolismo , Células-Tronco/metabolismoRESUMO
Exosomes are small membrane-bound vesicles that carry biological macromolecules from the site of production to target sites either in the microenvironment or at distant sites away from the origin. Exosomal content of cells varies with the cell type that produces them as well as environmental factors that alter the normal state of the cell such as viral infection. Human DNA and RNA viruses alter the composition of host proteins as well as incorporate their own viral proteins and other cargo into the secreted exosomes. While numerous viruses can infect various cell types of the CNS and elicit damaging neuropathologies, few have been studied for their exosomal composition, content, and function on recipient cells. Therefore, there is a pressing need to understand how DNA and RNA viral infections in CNS control exosomal release. Some of the more recent studies including HIV-1, HTLV-1, and EBV-infected B cells indicate that exosomes from these infections contain viral miRNAs, viral transactivators, and a host of cytokines that can control the course of infection. Finally, because exosomes can serve as vehicles for the cellular delivery of proteins and RNA and given that the blood-brain barrier is a formidable challenge in delivering therapeutics to the brain, exosomes may be able to serve as ideal vehicles to deliver protein or RNA-based therapeutics to the brain.
Assuntos
Viroses do Sistema Nervoso Central/patologia , Viroses do Sistema Nervoso Central/virologia , Exossomos/patologia , Exossomos/virologia , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por HTLV-I/patologia , Infecções por HTLV-I/virologia , Humanos , RNA ViralRESUMO
Chronic liver diseases are caused by hepatic viral infection, chemicals, and metabolic stress. The protein Grb2-associated binder 1 (Gab1) binds to various growth factor receptors, and triggers cell differentiation/survival signaling pathways. To identify signaling molecules involved in the progression of liver diseases, we performed reverse-phase protein microarray (RPMA)-based screening of hepatocytes isolated from humanized mice after acute HCV infection. Acute viral infection in humanized liver mice significantly decreased the level of hepatocyte p-Gab1. Moreover, hepatoma cells upon HCV infection decreased Gab1 mRNA at later times of infection (D3 to D5) and p-Gab1 level was inversely related to the production of TGF-ß. In contrast, the level of p-Gab1 was increased in CCL4-induced fibrotic liver. Hepatoma cells showed elevation of p-Gab1, along with an increase in STAT3 and ERK activation, upon treatment with HGF (ligand of HGF receptor/c-Met) and CCL4. In Gab1 knockdown hepatoma cells, cell proliferative signaling activity was reduced but the level of activated caspase-3 was increased. These findings suggest that hepatocyte Gab1 expression may play a role in promoting liver fibrosis progression by triggering ERK activation and inhibiting apoptosis. It implies that the Gab1-mediated signaling pathway would be a promising therapeutic target to treat chronic liver diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proliferação de Células , Fator de Crescimento de Hepatócito , Hepatócitos , Cirrose Hepática , Proteínas Proto-Oncogênicas c-met , Transdução de Sinais , Animais , Hepatócitos/metabolismo , Hepatócitos/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Linhagem Celular Tumoral , Hepatite C/metabolismo , Hepatite C/patologia , Hepatite C/complicaçõesRESUMO
The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.
Assuntos
Proteínas de Choque Térmico HSP90 , Complexo de Endopeptidases do Proteassoma , Proteólise , Vírus da Febre do Vale do Rift , Replicação Viral , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/genética , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Genoma Viral , Humanos , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , Interações Hospedeiro-Patógeno , Proteínas Virais/metabolismo , Proteínas Virais/genética , Transcrição Gênica , Febre do Vale de Rift/virologia , Febre do Vale de Rift/metabolismo , Linhagem CelularRESUMO
Recent findings have highlighted potential diagnostic and prognostic values of extracellular vesicles (EVs) that contain mitochondrial derived components for neurological disorders. Furthermore, functional influences of vesicles carrying mitochondrial components have been reported. In particular, this includes indications of crosstalk with mitophagy to influence progression of various CNS disorders. In this mini-review, we discuss the current state of knowledge about this intriguing class of vesicles in neurological disorders of the CNS, and outline the lacunae and thus scope of further development in this fascinating field of study.
RESUMO
BACKGROUND: The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has resulted in the infection of over 128 million people and has caused over 2.8 million deaths as of April 2021 in more than 220 countries and territories. Currently, there is no effective treatment for COVID-19 to reduce mortality. We investigated the potential anti-coronavirus activities from an oral liquid of traditional medicine, Respiratory Detox Shot (RDS), which contains mostly herbal ingredients traditionally used to manage lung diseases. RESULTS: Here we report that RDS inhibited the infection of target cells by lenti-SARS-CoV, lenti-SARS-CoV-2, and hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudoviruses, and by infectious SARS-CoV-2 and derived Ha-CoV-2 variants including B.1.1.7, B.1.351, P.1, B.1.429, B.1.2, B.1.494, B.1.1.207, B.1.258, and B.1.1.298. We further demonstrated that RDS directly inactivates the infectivity of SARS-CoV-2 virus particles. In addition, we found that RDS can also block the infection of target cells by Influenza A virus. CONCLUSIONS: These results suggest that RDS may broadly inhibit the infection of respiratory viruses.
RESUMO
BACKGROUND: Although multiple studies have demonstrated a role for exosomes during virus infections, our understanding of the mechanisms by which exosome exchange regulates immune response during viral infections and affects viral pathogenesis is still in its infancy. In particular, very little is known for cytoplasmic single-stranded RNA viruses such as SARS-CoV-2 and Rift Valley fever virus (RVFV). We have used RVFV infection as a model for cytoplasmic single-stranded RNA viruses to address this gap in knowledge. RVFV is a highly pathogenic agent that causes RVF, a zoonotic disease for which no effective therapeutic or approved human vaccine exist. RESULTS: We show here that exosomes released from cells infected with RVFV (designated as EXi-RVFV) serve a protective role for the host and provide a mechanistic model for these effects. Our results show that treatment of both naïve immune cells (U937 monocytes) and naïve non-immune cells (HSAECs) with EXi-RVFV induces a strong RIG-I dependent activation of IFN-B. We also demonstrate that this strong anti-viral response leads to activation of autophagy in treated cells and correlates with resistance to subsequent viral infection. Since we have shown that viral RNA genome is associated with EXi-RVFV, RIG-I activation might be mediated by the presence of packaged viral RNA sequences. CONCLUSIONS: Using RVFV infection as a model for cytoplasmic single-stranded RNA viruses, our results show a novel mechanism of host protection by exosomes released from infected cells (EXi) whereby the EXi activate RIG-I to induce IFN-dependent activation of autophagy in naïve recipient cells including monocytes. Because monocytes serve as reservoirs for RVFV replication, this EXi-RVFV-induced activation of autophagy in monocytes may work to slow down or halt viral dissemination in the infected organism. These findings offer novel mechanistic insights that may aid in future development of effective vaccines or therapeutics, and that may be applicable for a better molecular understanding of how exosome release regulates innate immune response to other cytoplasmic single-stranded RNA viruses.
RESUMO
The intense effort of investigators, in particular during the past decade, has highlighted the importance of extracellular vesicles (EVs) such as exosomes in regulating both innate and adaptive immunity in the course of a variety of infections, with clear implications for development of novel vaccines, therapeutics, and diagnostics. Current and future efforts now need to focus strongly on teasing apart the intricate and complex molecular mechanisms that operate during EV regulation of immunity. In this review, we discuss recent advances that bear on our current understanding of how EVs, including exosomes, can contribute to the innate immune functions of microglia within the central nervous system (CNS), and we also highlight future important mechanistic questions that need to be addressed. In particular, recent findings that highlight the crosstalk between autophagy and exosome pathways and their implications for innate immune functions of microglia will be presented. Microglial activation has been shown to play a key role in neuroAIDS, a neuro-infectious disease for which the importance of exosome functions, including exosome-autophagy interplay, has been reported. The importance of exosomes and exosome-autophagy crosstalk involving microglia has also been shown for the Parkinson's disease (PD), a neurodegenerative disease that is thought to be linked with immune dysfunction and involve infectious agents as trigger. Considering the accumulation of recent findings and the vibrancy of the EV field, we anticipate that future studies will continue to have a deep impact on our understanding of the CNS pathologies that are influenced by the functions of microglia and of the infectious disease mechanisms in general. Graphical Abstract.
Assuntos
Sistema Nervoso Central/imunologia , Sistema Nervoso Central/fisiologia , Vesículas Extracelulares/fisiologia , Microglia/imunologia , Microglia/fisiologia , Animais , Comunicação Celular , Vesículas Extracelulares/metabolismo , Humanos , Imunidade Celular , Doenças NeurodegenerativasRESUMO
Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0â»8.0), over a broad temperature range (4â»55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.
Assuntos
Fagos Bacilares/enzimologia , Bacillus/virologia , Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Fagos Bacilares/classificação , Fagos Bacilares/genética , Domínio Catalítico , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/farmacologia , Estabilidade Enzimática , Especificidade de Hospedeiro , Modelos Moleculares , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target.
Assuntos
Antivirais/farmacologia , Replicação do DNA/fisiologia , Indóis/farmacologia , Proteína Fosfatase 1/metabolismo , Vírus da Febre do Vale do Rift/enzimologia , Vírus da Febre do Vale do Rift/fisiologia , Ureia/análogos & derivados , Replicação Viral/fisiologia , Animais , Antivirais/química , Antivirais/uso terapêutico , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Genoma Viral/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Viral/biossíntese , RNA Viral/efeitos dos fármacos , Febre do Vale de Rift/tratamento farmacológico , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Vírus da Febre do Vale do Rift/genética , Ureia/farmacologia , Células Vero , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Virulência , Replicação Viral/efeitos dos fármacosRESUMO
Rift Valley Fever Virus (RVFV) is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent human immunodeficiency virus-1 (HIV-1) and human T-cell lymphotropic virus-1 (HTLV-1) infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-κB pathway, leading to cell proliferation, and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV). These clones contained normal markers (i.e., CD63) for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome). The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T-cells and monocytic cells) showed drastic rate of apoptosis through PARP cleavage and caspase 3 activation from some but not all exosome enriched preparations. Collectively, these data suggest that exosomes from RVFV infected cells alter the dynamics of the immune cells and may contribute to pathology of the viral infection.
RESUMO
Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts.
RESUMO
Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.
RESUMO
Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3ß suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.
RESUMO
Exosomes have recently been classified as the newest family members of 'bioactive vesicles' that function to promote intercellular communication. Long ignored and thought to be only a mechanism by which cellular waste is removed, exosomes have garnered a huge amount of interest in recent years as their critical functions in maintaining homeostasis through intercellular communication and also in different types of diseases have been demonstrated. Many groundbreaking studies of exosome functions have been performed in the cancer field and the infectious disease areas of study, revealing the importance and also the fascinating complexity of exosomal packaging, targeting, and functions. Selective packaging of exosomes in response to the type of infection, exosomal modulation of the immune response and host signaling pathways, exosomal regulation of pathogen spread, and effects of exosomes on the degree of pathogenesis have all been well documented. In this review, we provide a synthesis of the current understanding of the role of exosomes during infections caused by human pathogens and discuss the implications of these findings for a better understanding of pathogenic mechanisms and future therapeutic and diagnostic applications.
Assuntos
Comunicação Celular , Fenômenos Fisiológicos Celulares , Doenças Transmissíveis/patologia , Doenças Transmissíveis/fisiopatologia , Exossomos/metabolismo , Humanos , Modelos BiológicosRESUMO
Rift Valley fever virus (RVFV) infection is often associated with pronounced liver damage. Previously, our studies revealed altered host phospho-signaling responses (NFκB, MAPK and DNA damage responses) in RVFV infected epithelial cells that correlated with a cellular stress response. Here, we report that RVFV infection of liver cells leads to an increase in reactive oxygen species (ROS). Our data suggests the presence of the viral protein NSs in the mitochondria of infected cells, hence contributing to early increase in ROS. Increased ROS levels correlated with activation of NFκB (p65) and p53 responses, which in conjunction with infection, was also reflected as macromolecular rearrangements observed using size fractionation of protein lysates. Additionally, we documented an increase in cytokine expression and pro-apoptotic gene expression with infection, which was reversed with antioxidant treatment. Collectively, we identified ROS and oxidative stress as critical contributors to apoptosis of liver cells during RVFV infection.