RESUMEN
Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.
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Bioingeniería , Lentivirus , Proteínas de la Membrana , Músculo Esquelético , Distrofia Muscular de Duchenne , Animales , Ratones , Fusión Celular , Fusión de Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Músculo Esquelético/virología , Bioingeniería/métodos , Distrofia Muscular de Duchenne/terapia , Modelos Animales de Enfermedad , Tropismo Viral , Lentivirus/genéticaRESUMEN
Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (µDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Pruebas de Neutralización/métodos , SARS-CoV-2/inmunología , Adulto , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dominios Proteicos/inmunología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular New Jersey/inmunologíaRESUMEN
We describe a fatal case of multisystem inflammatory syndrome in an adult with onset 22 days after a second dose of mRNA coronavirus disease vaccine. Serologic and clinical findings indicated severe acute respiratory syndrome coronavirus 2 infection occurred before vaccination. The immunopathology of this syndrome, regardless of vaccination status, remains poorly understood.
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COVID-19 , Adulto , Vacunas contra la COVID-19 , Humanos , SARS-CoV-2 , Síndrome , VacunaciónRESUMEN
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is ongoing and has shown the community that flexible methods for rapidly identifying and screening candidate antivirals are needed. Assessing virus-neutralizing activity of human serum to monitor population immunity and response to infection and vaccination is key to pandemic control. We developed a virus neutralization platform strategy that relies only on bioinformatic and genetic information of the virus of interest. The platform uses viral envelope glycoprotein cDNAs to set up an assay that mimics multicycle infection but is safe and, therefore, amenable to biosafety level 2 (BSL2) conditions for viruses that require BSL3 facilities (e.g., SARS-CoV-1 and SARS-CoV-2). As a complement to this platform, we present a new cell-based immunofluorescent (CBI) assay that uses SARS-CoV-2 spike protein (S)-expressing cells to accurately measure the neutralization potential of human sera and is readily adaptable to variants of concern. These methods should be useful additions to the tools for assessing antiviral immunity, whether acquired via natural infection or vaccines. IMPORTANCE Assays for rapid biosafety level 2 (BSL2) evaluation of neutralizing properties of antibodies acquired via natural infection or through vaccination is urgently needed. Here, we propose a combinatorial approach in which sera are screened for SARS-CoV-2 spike protein (S) binding using a cell-based immunofluorescent (CBI) assay, and positive samples are further evaluated in a pseudotyped viral multicycle infection-mimicking protocol under BSL2 conditions.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , COVID-19/virología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Pruebas de Neutralización/métodos , Pandemias/prevención & control , Células VeroRESUMEN
Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies against the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological ELISA using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 reverse transcription PCR-confirmed, SARS-CoV-2-hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies against the 2 different viral proteins showed a moderate correlation. Antibodies against N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.9% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD-binding antibodies exhibited neutralizing capacity, but only 74% of N-protein-positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein-binding antibodies does not always correlate with presence of S-RBD-neutralizing antibodies and caution against the extensive use of N-protein-based serology testing for determination of potential COVID-19 immunity.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Betacoronavirus/fisiología , Infecciones por Coronavirus , Nucleocápside/inmunología , Pandemias , Neumonía Viral , Glicoproteína de la Espiga del Coronavirus/inmunología , Inmunidad Adaptativa/inmunología , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/terapia , Infecciones por Coronavirus/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Neumonía Viral/inmunología , Neumonía Viral/terapia , Neumonía Viral/virología , Unión Proteica , SARS-CoV-2 , Sensibilidad y Especificidad , Seroconversión , Pruebas Serológicas/métodosRESUMEN
The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches.
RESUMEN
OBJECTIVES: The bio-field array is a device that generates a dielectrophoretic electromagnetic field when placed in a hypotonic saline solution and a direct current of approximately 3 A is applied. It is known that cell physiology is guided by bioelectrical properties, and there is a significant growth inhibition in cancerous (MDA-MB-231) cells that are grown in media that has been reconstituted with the saline that has been exposed to the bio-field array direct current dielectrophoretic electromagnetic field, alternatively there is no growth inhibition noted in noncancerous cells (MCF-10A) when grown in the bio-field array direct current dielectrophoretic electromagnetic field treated versus control media. METHODS: To examine the basis for selective growth inhibition in human breast carcinoma, we employed cell death assays, cell cycle assays, microarray analysis and reverse transcription-quantitative polymerase chain reaction. RESULTS: We found a large transcriptional reprogramming in the cell lines and of the genes affected, those involved in endoplasmic reticulum stress and the unfolded protein response pathways showed some of the most dramatic changes. Cancerous cells grown in media that has been reconstituted with a hypotonic saline solution that has been exposed to the bio-field array direct current dielectrophoretic electromagnetic field show a significant and strong upregulation of the apoptotic arms of the unfolded protein response while the noncancerous cells show a decrease in endoplasmic reticulum stress via microarray analyses and reverse transcription-quantitative polymerase chain reaction. CONCLUSION: The bio-field array shows potential to initiate apoptosis in cancerous cells while relieving cell stress in noncancerous cells in vitro. These studies lay a foundation for nurses to conduct future in vivo models for the possible development of future adjunct treatments in chronic disease.
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To evaluate the role of cytoplasmic domains of membrane-spanning proteins in directing trafficking through the secretory pathway, we generated fluorescently tagged VSV G tsO45 with either the native G tail (G) or a cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE)). We previously showed that these two proteins progressed through the Golgi with distinct kinetics. To investigate the basis for the differential sorting of G and G(AE), we analyzed the role of several Golgi-associated small GTP-binding proteins and found that Rab43 differentially regulated their transport through the Golgi. We show that the expression of GFP-Rab43 arrested the anterograde transport of G(AE) in a Rab43-positive medial Golgi compartment. GFP-Rab43 expression also inhibited the acquisition of endoH-resistant sugars and the surface delivery of G(AE), as well as the surface delivery of the AE1-4 anion exchanger. In contrast, GFP-Rab43 expression did not affect the glycosylation or surface delivery of G. Unexpectedly, down-regulation of endogenous Rab43 using small interfering RNA resulted in an increase in the accumulation of G(AE) on the cell surface while having minimal effect on the surface levels of G. Our data demonstrate that Rab43 regulates the sorting of a subset of membrane-spanning cargo as they progress through the medial Golgi.
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Aparato de Golgi/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Vías SecretorasRESUMEN
There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.
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Virus ARN/inmunología , Vesiculovirus/genética , Vacunas Virales/inmunología , Animales , Humanos , Vacunas Sintéticas/inmunologíaRESUMEN
Melanoma risk is increased in patients with mutations of melanocortin 1 receptor (MC1R) yet the basis for the increased risk remains unknown. Here we report in vivo evidence supporting a critical role for MC1R in regulating melanoma tumor growth and determining overall survival time. Inhibition of MC1R by its physiologically relevant competitive inhibitor, agouti signaling protein (ASIP), reduced melanin synthesis and morphological heterogeneity in murine B16-F10 melanoma cells. In the lungs of syngeneic C57BL/6 mice, mCherry-marked, ASIP-secreting lung tumors inhibited MC1R on neighboring tumors lacking ASIP in a dose dependent manner as evidenced by a proportional loss of pigment in tumors from mice injected with 1:1, 3:1 and 4:1 mixtures of parental B16-F10 to ASIP-expressing tumor cells. ASIP-expressing B16-F10 cells formed poorly pigmented tumors in vivo that correlated with a 20% longer median survival than those bearing parental B16-F10 tumors (p=0.0005). Mice injected with 1:1 mixtures also showed survival benefit (p=0.0054), whereas injection of a 4:1 mixture showed no significant difference in survival. The longer survival time of mice bearing ASIP-expressing tumors correlated with a significantly slower growth rate than parental B16-F10 tumors as judged by quantification of numbers of tumors and total tumor load (p=0.0325), as well as a more homogeneous size and morphology of ASIP-expressing lung tumors. We conclude that MC1R plays an important role in regulating melanoma growth and morphology. Persistent inhibition of MC1R provided a significant survival advantage resulting in part from slower tumor growth, establishing MC1R as a compelling new molecular target for metastatic melanoma.
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Proteína de Señalización Agouti/metabolismo , Melanoma Experimental/patología , Receptor de Melanocortina Tipo 1/antagonistas & inhibidores , Proteína de Señalización Agouti/genética , Animales , Línea Celular Tumoral , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Bioelectrodynamics is an interdisciplinary subject that offers a pathway for nursing to develop a new patient care strategy in health care. The application of bioenergy to living organisms has the potential to advance medical science in the areas of prevention, cancer, wound care, pain, and many other chronic diseases.
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Salud Holística , Enfermería Holística/organización & administración , Comunicación Interdisciplinaria , Rol de la Enfermera , Grupo de Atención al Paciente/organización & administración , Biotecnología , Enfermería Holística/métodos , Humanos , Terapias Mente-CuerpoRESUMEN
To investigate the role of cytoplasmic sequences in directing transmembrane protein trafficking through the Golgi, we analyzed the sorting of VSV tsO45 G fusions with either the native G cytoplasmic domain (G) or an alternative cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE) ). At restrictive temperature G(AE) and G accumulated in the ER, and upon shifting the cells to permissive temperature both proteins folded and underwent transport through the Golgi. However, G(AE) and G did not form hetero-oligomers upon the shift to permissive temperature and they progressed through the Golgi with distinct kinetics. In addition, the transport of G through the proximal Golgi was Arf1 and COPI-dependent, while G(AE) progression through the proximal Golgi was Arf1 and COPI-independent. Although Arf1 did not regulate the sorting of G(AE) in the cis-Golgi, Arf1 did regulate the exit of G(AE) from the TGN. The trafficking of G(AE) through the Golgi was similar to that of the native AE1-4 anion exchanger, in that the progression of both proteins through the proximal Golgi was Arf1-independent, while both required Arf1 to exit the TGN. We propose that the differential recognition of cytosolic signals in membrane-spanning proteins by the Arf1-dependent sorting machinery may influence the rate at which cargo progresses through the Golgi.
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Factor 1 de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Transporte de Proteínas/fisiología , Transducción de Señal/fisiología , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Células COS , Línea Celular , Pollos , Chlorocebus aethiops , Proteína Coat de Complejo I/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Perros , Retículo Endoplásmico/metabolismo , Cinética , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína/fisiologíaRESUMEN
BACKGROUND: Nipah virus (NiV) is a highly pathogenic zoonotic agent in the family Paramyxoviridae that is maintained in nature by bats. Outbreaks have occurred in Malaysia, Singapore, India, and Bangladesh and have been associated with 40 to 75% case fatality rates. There are currently no vaccines or postexposure treatments licensed for combating human NiV infection. METHODS AND RESULTS: Four groups of ferrets received a single vaccination with different recombinant vesicular stomatitis virus vectors expressing: Group 1, control with no glycoprotein; Group 2, the NiV fusion protein (F); Group 3, the NiV attachment protein (G); and Group 4, a combination of the NiV F and G proteins. Animals were challenged intranasally with NiV 28 days after vaccination. Control ferrets in Group 1 showed characteristic clinical signs of NiV disease including respiratory distress, neurological disorders, viral load in blood and tissues, and gross lesions and antigen in target tissues; all animals in this group succumbed to infection by day 8. Importantly, all specifically vaccinated ferrets in Groups 2-4 showed no evidence of clinical illness and survived challenged. All animals in these groups developed anti-NiV F and/or G IgG and neutralizing antibody titers. While NiV RNA was detected in blood at day 6 post challenge in animals from Groups 2-4, the levels were orders of magnitude lower than animals from control Group 1. CONCLUSIONS: These data show protective efficacy against NiV in a relevant model of human infection. Further development of this technology has the potential to yield effective single injection vaccines for NiV infection.
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Portadores de Fármacos , Vectores Genéticos , Infecciones por Henipavirus/prevención & control , Virus Nipah/inmunología , Vacunación/métodos , Vesiculovirus/genética , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Hurones , Inmunoglobulina G/sangre , Virus Nipah/genética , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NFκB signaling pathway, the inhibitor of κB kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NFκB-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.
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Amidas/farmacología , Antivirales/farmacología , Glioma/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , Imidazoles/farmacología , Interferón Tipo I/farmacología , Quinoxalinas/farmacología , Tiofenos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Efecto Citopatogénico Viral/efectos de los fármacos , Virus de la Encefalomiocarditis/efectos de los fármacos , Virus de la Encefalomiocarditis/fisiología , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/genética , Glioma/virología , Humanos , Quinasa I-kappa B/metabolismo , Interferón Tipo I/inmunología , Interleucina-8/genética , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
Emerging viruses including Nipah, Hendra, Lujo, and Junin viruses have enormous potential to spread rapidly. Nipah virus, after emerging as a zoonosis, has also evolved the capacity for human-to-human transmission. Most of the diseases caused by these pathogens are untreatable and require high biocontainment conditions. Universal methods for rapidly identifying and screening candidate antivirals are urgently needed. We have developed a modular antiviral platform strategy that relies on simple bioinformatic and genetic information about each pathogen. Central to this platform is the use of envelope glycoprotein cDNAs to establish multi-cycle replication systems under BSL2 conditions for viral pathogens that normally require BSL3 and BSL4 facilities. We generated monoclonal antibodies against Nipah G by cDNA immunization in rats, and we showed that these antibodies neutralize both Nipah and Hendra live viruses. We then used these effective Henipavirus inhibitors to validate our screening strategy. Our proposed strategy should contribute to the response capability for emerging infectious diseases, providing a way to initiate antiviral development immediately upon identifying novel viruses.
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Biología Computacional/métodos , Virus Hendra/metabolismo , Virus Nipah/metabolismo , Animales , Anticuerpos Neutralizantes/química , Antivirales/farmacología , Chlorocebus aethiops , ADN Complementario/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Humanos , Inmunoterapia/métodos , Ratas , Tecnología Farmacéutica/métodos , Células Vero , Replicación ViralRESUMEN
Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.
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Vectores Genéticos/inmunología , Epítopos Inmunodominantes/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Antígenos Virales/inmunología , Brefeldino A/inmunología , Células Cultivadas , Cricetinae , Citomegalovirus/inmunología , Replicación del ADN/inmunología , Células Dendríticas/inmunología , Vectores Genéticos/genética , Humanos , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Monocitos/inmunología , Orthomyxoviridae/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas Virales/genética , Proteínas Virales/inmunología , Replicación Viral , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells. While fluorescent protein fusions (e.g. viral proteins fused to GFP) have been used, the labeling of viral proteins that contain a small tetracysteine (tc) tag with biarsenical fluorescent compounds (e.g. FlAsH, ReAsH, Lumio-x) offers several advantages over conventional xFP-fusion constructs. This article describes methods for generating fluorescently labeled viruses encoding tc-tagged proteins that are suitable for the study of virus entry in live cells by fluorescence microscopy. Critical parameters required to quantify fluorescence signals from the labeled, tc-tagged proteins in individual virus particles during the entry process and the subsequent fate of the labeled viral proteins after virus uncoating are also described.
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Microscopía Confocal/métodos , Oligopéptidos/metabolismo , Análisis de la Célula Individual/métodos , Vesiculovirus/fisiología , Proteínas Estructurales Virales/metabolismo , Internalización del Virus , Secuencia de Aminoácidos , Animales , Técnicas de Cultivo de Célula , Células Cultivadas , Cricetinae , Fluoresceínas/química , Colorantes Fluorescentes/química , Datos de Secuencia Molecular , Compuestos Organometálicos/química , Proteínas Recombinantes de Fusión , Coloración y Etiquetado/métodos , Vesiculovirus/química , Vesiculovirus/metabolismo , Proteínas Estructurales Virales/químicaRESUMEN
To study the contribution of the protease-sensitive loop of the VSV M protein in virus assembly we recovered recombinant VSV (rVSV) with mutations in this region and examined virus replication. Mutations in the highly conserved LXD motif (aa 123-125) resulted in reduced virion budding, reduced virus titers and enhanced M protein exchange with M-ribonucleocapsid complexes (M-RNPs), suggesting that the mutant M proteins were less tightly associated with RNP skeletons. In addition, viral protein synthesis began to decrease at 4h post-infection (hpi) and was reduced by ~80% at 8 hpi for the mutant rVSV-D125A. The reduced protein synthesis was not due to decreased VSV replication or transcription; however, translation of a reporter gene with an EMCV IRES was not reduced, suggesting that cap-dependent, but not cap-independent translation initiation was affected in rVSV-D125A infected cells. These results indicate that the LXD motif is involved in both virus assembly and VSV protein translation.