RESUMO
Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been recognized as promising cytotherapeutics due to their demonstrated immunomodulatory effects in various preclinical models. The immunomodulatory capabilities of EVs stem from the proteins and genetic materials they carry from parent cells, but the cargo contents of EVs are significantly influenced by MSC tissues and donors, cellular age and culture conditions, resulting in functional variations. However, there are no surrogate assays available to validate the immunomodulatory potency of MSC-EVs before in vivo administration. In previous work, we discovered that microcarrier culture conditions enhance the immunomodulatory function of MSC-EVs, as well as the levels of immunosuppressive molecules such as TGF-ß1 and let-7b in MSC-EVs. Building on these findings, we investigated whether TGF-ß1 levels in MSC-EVs could serve as a surrogate biomarker for predicting their potency in vivo. Our studies revealed a strong correlation between TGF-ß1 and let-7b levels in MSC-EVs, as well as their capacity to suppress IFN-γ secretion in stimulated splenocytes, establishing biopotency and surrogate assays for MSC-EVs. Subsequently, we validated MSC-EVs generated from monolayer cultures (ML-EVs) or microcarrier cultures (MC-EVs) using murine models of experimental autoimmune uveoretinitis (EAU) and additional in vitro assays reflecting the Mode of Action of MSC-EVs in vivo. Our findings demonstrated that MC-EVs carrying high levels of TGF-ß1 exhibited greater efficacy than ML-EVs in halting disease progression in mice with EAU as well as inducing apoptosis and inhibiting the chemotaxis of retina-reactive T cells. Additionally, MSC-EVs suppressed the MAPK/ERK pathway in activated T cells, with treatment using TGF-ß1 or let-7b showing similar effects on the MAPK/ERK pathway. Collectively, our data suggest that MSC-EVs directly inhibit the infiltration of retina-reactive T cells toward the eyes, thereby halting the disease progression in EAU mice, and their immunomodulatory potency in vivo can be predicted by their TGF-ß1 levels.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , Fator de Crescimento Transformador beta1 , Uveíte , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Camundongos , Uveíte/terapia , Uveíte/imunologia , Uveíte/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , MicroRNAs/metabolismo , Doenças Autoimunes/terapia , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Imunomodulação , Camundongos Endogâmicos C57BL , Humanos , FemininoRESUMO
BACKGROUND: Corneal transplantation is the most common transplant procedure worldwide. Despite immune and angiogenic privilege of the cornea, 50% to 70% of corneal transplants fail in high-risk recipients, primarily because of immune rejection. Therefore, it is crucial to identify predictive biomarkers of rejection to improve transplant survival. METHODS: In search for predictive biomarkers, we performed proteomics analysis of serum extracellular vesicles (EVs) in a fully major histocompatibility complex-mismatched (C57BL/6-to-BALB/c) murine corneal transplantation model, wherein 50% of transplants undergo rejection by day 28 following transplantation. RESULTS: Our time course study revealed a decrease in the number of serum EVs on day 1, followed by a gradual increase by day 7. A comparative analysis of proteomics profiles of EVs from transplant recipients with rejection (rejectors) and without rejection (nonrejectors) found a distinct enrichment of histocompatibility 2, Q region locus 2, which is a part of major histocompatibility complex-class I of donor C57BL/6 mice, in day 7 EVs of rejectors, compared with nonrejectors, syngeneic controls, or naïve mice. In contrast, serum amyloid A2, a protein induced in response to injury, was increased in day 7 EVs of nonrejectors. CONCLUSIONS: Our findings offer noninvasive EV-based potential biomarkers for predicting corneal allograft rejection or tolerance.
Assuntos
Biomarcadores , Transplante de Córnea , Vesículas Extracelulares , Rejeição de Enxerto , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteômica , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/diagnóstico , Animais , Vesículas Extracelulares/metabolismo , Biomarcadores/sangue , Proteômica/métodos , Camundongos , Sobrevivência de Enxerto , Modelos Animais de Doenças , Valor Preditivo dos Testes , MasculinoRESUMO
Extracellular vesicles (EVs) from allogeneic-tissue-derived mesenchymal stem cells (MSCs) are promising to improve Sjögren's syndrome (SS) treatment, but their application is hindered by high variations in and limited expandability of tissue MSCs. We derived standardized and scalable MSCs from iPS cells (iMSCs) and reported that EVs from young but not aging iMSCs (iEVs) inhibited sialadenitis onset in SS mouse models. Here, we aim to determine cellular mechanisms and optimization approaches of SS-inhibitory effects of iEVs. In NOD.B10.H2b mice at the pre-disease stage of SS, we examined the biodistribution and recipient cells of iEVs with imaging, flow cytometry, and qRT-PCR. Intravenously infused iEVs accumulated in the spleen but not salivary glands or cervical lymph nodes and were mainly taken up by macrophages. In the spleen, young but not aging iEVs increased M2 macrophages, decreased Th17 cells, and changed expression of related immunomodulatory molecules. Loading miR-125b inhibitors into aging iEVs significantly improved their effects on repressing sialadenitis onset and regulating immunomodulatory splenocytes. These data indicated that young but not aging iEVs suppress SS onset by regulating immunomodulatory splenocytes, and inhibiting miR-125b in aging iEVs restores such effects, which is promising to maximize production of effective iEVs from highly expanded iMSCs for future clinical application.
Assuntos
Vesículas Extracelulares , Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , MicroRNAs , Sialadenite , Síndrome de Sjogren , Camundongos , Animais , Síndrome de Sjogren/terapia , Síndrome de Sjogren/tratamento farmacológico , Baço/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Distribuição Tecidual , Camundongos Endogâmicos NOD , Sialadenite/terapia , Sialadenite/metabolismo , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Modelos Animais de DoençasRESUMO
Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.
Assuntos
Células-Tronco Mesenquimais , Animais , Antígenos HLA , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/farmacologia , Humanos , Células Matadoras Naturais , CamundongosRESUMO
Accumulating evidence indicates that mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) exhibit immunomodulatory effects by delivering therapeutic RNAs and proteins; however, the molecular mechanism underlying the EV-mediated immunomodulation is not fully understood. In this study, we found that EVs from early-passage MSCs had better immunomodulatory potency than did EVs from late-passage MSCs in T cell receptor (TCR)- or Toll-like receptor 4 (TLR4)-stimulated splenocytes and in mice with ocular Sjögren's syndrome. Moreover, MSC-EVs were more effective when produced from 3D culture of the cells than from the conventional 2D culture. Comparative molecular profiling using proteomics and microRNA sequencing revealed the enriched factors in MSC-EVs that were functionally effective in immunomodulation. Among them, manipulation of transforming growth factor ß1 (TGF-ß1), pentraxin 3 (PTX3), let-7b-5p, or miR-21-5p levels in MSCs significantly affected the immunosuppressive effects of their EVs. Furthermore, there was a strong correlation between the expression levels of TGF-ß1, PTX3, let-7b-5p, or miR-21-5p in MSC-EVs and their suppressive function. Therefore, our comparative strategy identified TGF-ß1, PTX3, let-7b-5p, or miR-21-5p as key molecules mediating the therapeutic effects of MSC-EVs in autoimmune disease. These findings would help understand the molecular mechanism underlying EV-mediated immunomodulation and provide functional biomarkers of EVs for the development of robust EV-based therapies.
Assuntos
Proteína C-Reativa/genética , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Componente Amiloide P Sérico/genética , Síndrome de Sjogren-Larsson/terapia , Fator de Crescimento Transformador beta1/genética , Animais , Proteína C-Reativa/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteômica , Inoculações Seriadas , Componente Amiloide P Sérico/metabolismo , Síndrome de Sjogren-Larsson/genética , Síndrome de Sjogren-Larsson/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Obesity has been associated with increased symptoms and mortality in influenza patients and impaired immune responses to the influenza vaccine. To date, however, there is no effective adjuvant to improve vaccine efficacy for the obese population. To address this issue, we generated a modified outer membrane vesicle with attenuated endotoxicity (fmOMV) and tested its adjuvant effect on the influenza vaccine in comparison with a squalene-based oil-in-water adjuvant (AddaVax) using a diet-induced obese (DIO) mouse model. Although coadministration of fmOMV did not affect neutralizing antibody (Ab) response, it preferentially induced IgG2c antibody response and significantly increased the vaccine-induced T cell response. More importantly, fmOMV conferred significant protection against homologous and heterologous influenza virus challenge, whereas AddaVax showed marginal protection irrespective of the strongest Ab and T cell responses in DIO mice. These results indicate that fmOMV improves the antigen-specific T cell response and the efficacy of an influenza vaccine, suggesting a potential influenza vaccine adjuvant for the obese population.
Assuntos
Adjuvantes Imunológicos , Vesículas Extracelulares/metabolismo , Imunogenicidade da Vacina , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Dieta , Modelos Animais de Doenças , Imunidade Celular , Vírus da Influenza A/imunologia , Masculino , Camundongos , Camundongos Obesos , Testes de Neutralização , Obesidade , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/mortalidadeRESUMO
Dengue virus (DENV) is a major infectious viral pathogen that affects millions of individuals worldwide every year, causing a potentially fatal syndrome, while no commercial antiviral drugs are yet available. To develop an antiviral against dengue fever, it is necessary to understand the relationship between DENV and host cells, which could provide a basis for viral dynamics and identification of inhibitory drug targets. In this study, we designed DiD-loaded and BODIPY-ceramide-encapsulated DENV-polymersome hybrid nanovesicles (DENVSomes) prepared by an extrusion method, which trigger red fluorescence in the endosome and green in the Golgi. DENVSome monitors the dynamics of host cell-virus interaction and tracking in living cells with novel state-of-the-art imaging technologies that show images at high resolution. Also, DENVSome can be exploited to screen whether candidate antiviral drugs interact with DENVs. Consequently, we successfully demonstrated that DENVSome is an efficient tool for tracking and unraveling the mechanisms of replication and drug screening for antiviral drugs of DENV.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Compostos de Boro/química , Rastreamento de Células , Dengue/virologia , Vírus da Dengue/química , Vírus da Dengue/fisiologia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Corantes Fluorescentes/química , Humanos , Nanopartículas/química , Replicação Viral/efeitos dos fármacosRESUMO
Influenza A virus (IAV) poses a constant worldwide threat to human health. Although conventional vaccines are available, their protective efficacy is type or strain specific, and their production is time-consuming. For the control of an influenza pandemic in particular, agents that are immediately effective against a wide range of virus variants should be developed. Although pretreatment of various Toll-like receptor (TLR) ligands have already been reported to be effective in the defense against subsequent IAV infection, the efficacy was limited to specific subtypes, and safety concerns were also raised. In this study, we investigated the protective effect of an attenuated bacterial outer membrane vesicle -harboring modified lipid A moiety of lipopolysaccharide (fmOMV) against IAV infection and the underlying mechanisms. Administration of fmOMV conferred significant protection against a lethal dose of pandemic H1N1, PR8, H5N2, and highly pathogenic H5N1 viruses; this broad antiviral activity was dependent on macrophages but independent of neutrophils. fmOMV induced recruitment and activation of macrophages and elicited type I IFNs. Intriguingly, fmOMV showed a more significant protective effect than other TLR ligands tested in previous reports, without exhibiting any adverse effect. These results show the potential of fmOMV as a prophylactic agent for the defense against influenza virus infection.
Assuntos
Membrana Externa Bacteriana/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Lipídeo A/imunologia , Macrófagos/imunologia , Infecções por Orthomyxoviridae/imunologia , Vesículas Secretórias/imunologia , Animais , Escherichia coli/genética , Feminino , Humanos , Interferon Tipo I/metabolismo , Ligantes , Lipídeo A/genética , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptores Toll-Like/agonistasRESUMO
Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes.
RESUMO
Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103+ dendritic cells in the mediastinal lymph nodes was significantly increased after fmOMV co-administration. Notably, the mice co-immunized with fmOMV showed a significantly higher protection rate against challenge with a lethal dose of homologous or heterologous influenza viruses without adverse effects. These results show the potential of fmOMV as an effective mucosal adjuvant for intranasal vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vesículas Extracelulares , Vacinas contra Influenza/imunologia , Lipídeo A/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Orthomyxoviridae/imunologia , Administração Intranasal , Animais , Anticorpos Antivirais/sangue , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Imunoglobulina A/análise , Vacinas contra Influenza/administração & dosagem , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Mucosa/imunologia , Análise de Sobrevida , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
Three major classes of retroviral restriction factors (APOBEC3G, Tetherin, and TRIM5α) have been identified in mammals. Restriction factors are cellular proteins that are able to limit viral replication by targeting specific steps of the viral life cycle. To evaluate which restriction factor is the most effective to inhibit the replication of porcine endogenous retroviruses (PERVs), the antiviral activity of each restriction factor was compared. In pseudotype assay, the antiviral activity of human tetherin against PERV pseudotype was slightly weaker than that of human APOBEC3G (hA3G). A combination of tetherin and hA3G was more potent than each individual restriction factor. We questioned whether a combination of tetherin and hA3G could also inhibit the spreading replication of PERV. In agreement with the pseudotype assay, two restriction factors inhibit infectious PERV replication in a spreading infection. In this study, hA3G could strongly inhibit the replication of PERV, but tetherin modestly restricted it. Based on these results, we concluded that a combination of tetherin and hA3G is the most effective way to restrict PERV. A combination of different restriction factors will encourage the development of a new approach to treat retroviral disease.
Assuntos
Antígenos CD/imunologia , Antivirais/imunologia , Proteínas de Transporte/imunologia , Citidina Desaminase/imunologia , Retrovirus Endógenos/imunologia , Retrovirus Endógenos/fisiologia , Replicação Viral , Desaminase APOBEC-3G , Animais , Fatores de Restrição Antivirais , Linhagem Celular , Proteínas Ligadas por GPI/imunologia , Humanos , Suínos , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.
Assuntos
Carlavirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Lilium/virologia , Doenças das Plantas/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Carlavirus/genética , Carlavirus/imunologia , Clonagem Molecular , Folhas de Planta/virologia , CoelhosRESUMO
When full-length molecular clones of porcine endogenous retrovirus (PERV)-A and porcine endogenous retrovirus (PERV)-B were passaged on human cells, an increase in the length of the long terminal repeat (LTR) was reported. A 39-bp repeat box in the LTR U3 region was multimerized dynamically upon replication, acting as a viral enhancer element that contains binding sites for nuclear transcription factor NF-Y. To analyze the optimum number of 39-bp repeats for viral replication, molecular clones of PERV-A with one, two, three, and four 39-bp repeats were constructed. Each full-length PERV-A molecular clone contained a different number of 39-bp repeat boxes and was used to transfect human 293 cells, the relative transcriptional activity of the LTRs 48 h posttransfection was determined. PERV LTRs containing 3 copies of a 39-bp repeat showed the strongest promoter activity by real-time reverse transcription PCR in human 293 cell lines. Virions generated by the transfection of a provirus with 3 enhancer repeats replicated efficiently in human cells and 2.5×10(4)virion copies/µL were released. Although the transcriptional activity of all PERV-A LTRs was lower than 293 cells (293-PERV-PK-CIRCE) infected productively with PERV-A and PERV-B. It was found that 3 was the optimal number of 39-bp repeats for viral replication. This molecular clone with a higher replication capacity could be used to study the biology of porcine endogenous retrovirus by genetic approaches or in vivo infection experiments.
Assuntos
Retrovirus Endógenos/fisiologia , Sequências Repetidas Terminais , Replicação Viral , Animais , Sítios de Ligação , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular , Retrovirus Endógenos/genética , Humanos , Suínos , Transcrição Gênica , TransfecçãoRESUMO
Nude mice (BALB/c) were grafted with human 293 cells and PERV (porcine endogenous retrovirus)-IRES-EGFP (a packageable retroviral vector plasmid containing an internal ribosome entry site-enhanced green fluorescent protein)-producing pig PK15 cells in order to determine whether the pig cells could transmit PERV-IRES-EGFP to mice and human 293 cells in vivo. None of the transplanted human 293 cell lines were infected by PERV, but PCR analysis identified PERV-B provirus integration into both the heart and salivary gland of the inoculated nude mice. Our data indicate that hearts and salivary glands can be used to identify PERV-B receptors.
Assuntos
Retrovirus Endógenos/fisiologia , Gammaretrovirus/fisiologia , Infecções por Retroviridae/transmissão , Infecções por Retroviridae/veterinária , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Retrovirus Endógenos/genética , Retrovirus Endógenos/isolamento & purificação , Gammaretrovirus/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Infecções por Retroviridae/virologia , Suínos , Doenças dos Suínos/transmissão , Transplante HeterólogoRESUMO
Porcine endogenous retroviruses (PERVs) present a unique concern associated with xenotransplantation because they have been shown to infect certain human cells in vitro and it is also difficult to generate herds of pigs free of PERVs. A simple system for the production of high-titer MoMLV-PERV pseudotypes is reported; an EGFP-expressing replication-competent molecular clone that allows direct measurement of titer was also constructed. To improve the MLV-based retroviral vector system, a 2.1-kb PERV-B env product was amplified from PK-15 genomic DNA and cloned into the pCL-Eco retroviral vector. The titer of lacZ (PERV-B) from the 293 cells was about 1.0×10(4) CFU/ml. In contrast, the titer of lacZ (PERV-B) from a conventional murine retroviral vector (split genome) was found to be 1.2×10(2) CFU/ml when the PERV-B env expression vector was transfected into TELCeB6 cells, which harbor MFGnlslacZ and the gag-pol-expressing vector. In addition, an infectious PERV-A clone containing enhanced GFP (EGFP) by using a PCR-based method was developed. This EGFP-expressing PERV-A-IRES-EGFP molecular clone was found to be stable genetically on transfection in 293 cells.
Assuntos
Retrovirus Endógenos/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Coloração e Rotulagem/métodos , Suínos/virologia , Animais , Linhagem Celular , Instabilidade Genômica , Proteínas de Fluorescência Verde/genética , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.
Assuntos
Infecções por Retroviridae/virologia , Retroviridae/genética , Sus scrofa/virologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Reservatórios de Doenças/virologia , Humanos , Coreia (Geográfico) , Dados de Sequência Molecular , Mutação Puntual , Recombinação Genética , Retroviridae/química , Retroviridae/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
Retrovirus tropism can be restricted by host cell factors such as Fv1, TRIM5alpha, and Lv1 that inhibit infection by targeting the incoming viral capsid. The Fv1 gene inhibits murine leukemia virus infection in mice, but the precise mechanism of Fv1-mediated restriction is poorly understood. Our previous studies had demonstrated that Fv1-mediated viral tropism can be determined within the capsid protein at position 114 (Jung and Kozak. 2000. J. Virol. 74: 5385-7). To study the interaction between Fv1 and CA, we introduced amino acid substitution and deletion at this site in the N-tropic AKV capsid gene. The mutated two-LTR proviral DNAs were introduced into SC-1 cells by transfection. After transfection, cell supernatants collected from transfected cells were tested for host range susceptibility. The result indicated that substitution of amino acids did not alter tropism, but the deletion of 114His produced a virus with unusual tropism. The novel phenotype produced here failed to replicate in Fv1-expressing cells. This mutant virus showing such an extreme restriction pattern would be useful for studying the mechanism of Fv1- mediated restriction.
Assuntos
Produtos do Gene gag/metabolismo , Vírus da Leucemia Murina/fisiologia , Proteínas/metabolismo , Tropismo , Substituição de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Produtos do Gene gag/genética , Vírus da Leucemia Murina/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Proteínas/genética , Sequências Repetidas Terminais , Transfecção , Replicação ViralRESUMO
The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.