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1.
Cell Commun Signal ; 20(1): 4, 2022 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-34998412

RESUMEN

BACKGROUND: Research is revealing the complex coordination between cell signaling systems as they adapt to genetic and epigenetic changes. Tools to uncover these highly complex functional linkages will play an important role in advancing more efficacious disease treatments. Current tumor cell signal transduction research is identifying coordination between receptor types, receptor families, and transduction pathways to maintain tumor cell viability despite challenging tumor microenvironment conditions. METHODS: In this report, coactivated abnormal levels of signaling activity for c-Met and HER family receptors in live tumor cells were measured by a new clinical test to identify a subpopulation of breast cancer patients that could be responsive to combined targeted therapies. The CELsignia Multi-Pathway Signaling Function (CELsignia) Test uses an impedance biosensor to quantify an individual patient's ex vivo live tumor cell signaling response in real-time to specific HER family and c-Met co-stimulation and targeted therapies. RESULTS: The test identified breast tumors with hyperactive HER1, HER2, HER3/4, and c-Met coordinated signaling that express otherwise normal amounts of these receptors. The supporting data of the pre-clinical verification of this test included analyses of 79 breast cancer patients' cell response to HER and c-Met agonists. The signaling results were confirmed using clinically approved matching targeted drugs, and combinations of targeted drugs in addition to correlative mouse xenograft tumor response to HER and c-Met targeted therapies. CONCLUSIONS: The results of this study demonstrated the potential benefit of a functional test for identifying a subpopulation of breast cancer patients with coordinated abnormal HER and c-Met signaling for a clinical trial testing combination targeted therapy. Video Abstract.


Asunto(s)
Neoplasias de la Mama , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Receptor ErbB-2/metabolismo , Transducción de Señal , Microambiente Tumoral
2.
J Virol ; 94(6)2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-31896593

RESUMEN

Rotaviruses (RV) cause acute severe diarrhea in the absence of substantial intestinal inflammation. They are also highly infectious in their homologous host species. The replication capacity of RV in the small bowel is substantially due to its ability to inhibit different types of interferons (IFNs). Here, we found that during RV infection in vitro, both virus-infected and uninfected bystander cells resist STAT1 phosphorylation and interferon regulatory factor 7 (IRF7) induction in response to exogenous interferon (IFN). Functionally, cellular transcription in response to stimulation with IFN, but not intracellular double-stranded RNA (dsRNA), was inhibited by RV. Further, IFNAR1 stimulation during RV infection significantly repressed a set of virus-induced transcripts. Regulation of IFN signaling in vivo was studied in suckling mice using the highly infectious murine EW RV strain. Kinetic studies indicated that sustained EW RV replication and IFN induction in the small intestine are accompanied by significant decreases in IFN-stimulated transcripts. Lipopolysaccharide (LPS)-mediated intestinal damage, driven by STAT1-induced inflammation, was also prevented in EW RV-infected mice. Remarkably, by ectopically stimulating either IFNAR1 or IFNGR1 in EW RV-infected mice, we could eliminate several intestinal antiviral and inflammatory transcriptional responses to RV. In contrast to infection with homologous RV, infection with a STAT1-sensitive heterologous RV strain induced IFN-stimulated transcripts, inflammatory cytokines, and intestinal expression of STAT1-pY701. Finally, RV strain-specific STAT1 regulation also likely determines the intestinal activation of multiple caspases. The simian RRV strain, but not murine EW RV, uniquely triggers the cleavage of both extrinsic and intrinsic caspases (caspases 8, 9, and 3) in a STAT1-mediated manner. Collectively, our findings reveal efficient reprograming of multiple IFN receptors toward a negative-feedback mode of signaling, accompanied by suppression of IFN-mediated antiviral, apoptotic, and inflammatory functions, during natural RV intestinal infection.IMPORTANCE Rotavirus is a highly infectious pathogen that causes severe diarrhea. Replication of RV in the small intestine is restricted to homologous host species, and host range restriction is substantially determined by the interferon response. In this study, we demonstrate that during infection, RV bystander cells resist exogenous IFN-mediated STAT1 signaling and transcription. In a suckling mouse model, ectopically stimulating different intestinal interferon receptors during RV infection eliminates several innate and inflammatory antiviral responses. Different intestinal inflammatory cytokines were also suppressed by homologous RV, as was intestinal damage in response to endotoxin. The ability of RV to suppress IFN-mediated receptors likely impacts intestinal cell homeostasis, as the cleavage of multiple intestinal caspases during RV infection is mediated by the IFN-STAT1 signaling pathway. Together, our results provide a mechanism underlying both the remarkable interferon resistance of homologous RV and its ability to prevent substantial inflammatory damage to the small bowel.


Asunto(s)
Enfermedades Intestinales/metabolismo , Mucosa Intestinal/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Receptores de Interferón/metabolismo , Infecciones por Rotavirus/metabolismo , Rotavirus/metabolismo , Animales , Caspasas/metabolismo , Citocinas/metabolismo , Células HEK293 , Células HT29 , Humanos , Inflamación/metabolismo , Inflamación/patología , Inflamación/virología , Enfermedades Intestinales/patología , Enfermedades Intestinales/virología , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Ratones , Infecciones por Rotavirus/patología , Factor de Transcripción STAT1/metabolismo , Receptor de Interferón gamma
3.
J Virol ; 92(1)2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-29070687

RESUMEN

STAT1 phosphorylation in response to exogenous interferon (IFN) administration can be inhibited by rotaviral replication both in vitro and in vivo In addition many rotavirus strains are resistant to the actions of different IFN types. The regulation by rotaviruses (RVs) of antiviral pathways mediated by multiple IFN types is not well understood. In this study, we find that during infection in vitro and in vivo, RVs significantly deplete IFN type I, II, and III receptors (IFNRs). Regulation of IFNRs occurred exclusively within RV-infected cells and could be abrogated by inhibiting the lysosomal-endosomal degradation pathway. In vitro, IFNR degradation was conserved across multiple RV strains that differ in their modes of regulating IFN induction. In suckling mice, exogenously administered type I, II, or III IFN induced phosphorylation of STAT1-Y701 within intestinal epithelial cells (IECs) of suckling mice. Murine EW strain RV infection transiently activated intestinal STAT1 at 1 day postinfection (dpi) but not subsequently at 2 to 3 dpi. In response to injection of purified IFN-α/ß or -λ, IECs in EW-infected mice exhibited impaired STAT1-Y701 phosphorylation, correlating with depletion of different intestinal IFNRs and impaired IFN-mediated transcription. The ability of EW murine RV to inhibit multiple IFN types led us to test protection of suckling mice from endotoxin-mediated shock, an outcome that is dependent on the host IFN response. Compared to mortality in controls, mice infected with EW murine RV were substantially protected against mortality following parenteral endotoxin administration. These studies identify a novel mechanism of IFN subversion by RV and reveal an unexpected protective effect of RV infection on endotoxin-mediated shock in suckling mice.IMPORTANCE Antiviral functions of types I, II, and III IFNs are mediated by receptor-dependent activation of STAT1. Here, we find that RV degrades the types I, II, and III IFN receptors (IFNRs) in vitro In a suckling mouse model, RV effectively blocked STAT1 activation and transcription following injection of different purified IFNs. This correlated with significantly decreased protein expression of intestinal types I and II IFNRs. Recent studies demonstrate that in mice lipopolysaccharide (LPS)-induced lethality is prevented by genetic ablation of IFN signaling genes such as IFNAR1 and STAT1. When suckling mice were infected with RV, they were substantially protected from lethal exposure to endotoxin. These findings provide novel insights into the mechanisms underlying rotavirus regulation of different interferons and are likely to stimulate new research into both rotavirus pathogenesis and endotoxemia.


Asunto(s)
Intestinos/citología , Lipopolisacáridos/toxicidad , Receptores de Interferón/metabolismo , Infecciones por Rotavirus/metabolismo , Rotavirus/fisiología , Factor de Transcripción STAT1/metabolismo , Animales , Animales Lactantes , Células Cultivadas , Endosomas/metabolismo , Endotoxinas/toxicidad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Células HEK293 , Células HT29 , Humanos , Intestinos/virología , Lisosomas/metabolismo , Ratones , Fosforilación , Proteolisis , Receptores de Interferón/genética , Rotavirus/clasificación , Transducción de Señal , Replicación Viral
4.
PLoS Pathog ; 12(10): e1005929, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27706223

RESUMEN

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and ß-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated ß-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets ß-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote ß-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Interacciones Huésped-Parásitos/fisiología , Infecciones por Rotavirus/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Animales , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Inmunoprecipitación , Espectrometría de Masas , Proteómica/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección
6.
PLoS Pathog ; 12(4): e1005600, 2016 04.
Artículo en Inglés | MEDLINE | ID: mdl-27128797

RESUMEN

Type I (IFN-α/ß) and type III (IFN-λ) interferons (IFNs) exert shared antiviral activities through distinct receptors. However, their relative importance for antiviral protection of different organ systems against specific viruses remains to be fully explored. We used mouse strains deficient in type-specific IFN signaling, STAT1 and Rag2 to dissect distinct and overlapping contributions of type I and type III IFNs to protection against homologous murine (EW-RV strain) and heterologous (non-murine) simian (RRV strain) rotavirus infections in suckling mice. Experiments demonstrated that murine EW-RV is insensitive to the action of both types of IFNs, and that timely viral clearance depends upon adaptive immune responses. In contrast, both type I and type III IFNs can control replication of the heterologous simian RRV in the gastrointestinal (GI) tract, and they cooperate to limit extra-intestinal simian RRV replication. Surprisingly, intestinal epithelial cells were sensitive to both IFN types in neonatal mice, although their responsiveness to type I, but not type III IFNs, diminished in adult mice, revealing an unexpected age-dependent change in specific contribution of type I versus type III IFNs to antiviral defenses in the GI tract. Transcriptional analysis revealed that intestinal antiviral responses to RV are triggered through either type of IFN receptor, and are greatly diminished when receptors for both IFN types are lacking. These results also demonstrate a murine host-specific resistance to IFN-mediated antiviral effects by murine EW-RV, but the retention of host efficacy through the cooperative action by type I and type III IFNs in restricting heterologous simian RRV growth and systemic replication in suckling mice. Collectively, our findings revealed a well-orchestrated spatial and temporal tuning of innate antiviral responses in the intestinal tract where two types of IFNs through distinct patterns of their expression and distinct but overlapping sets of target cells coordinately regulate antiviral defenses against heterologous or homologous rotaviruses with substantially different effectiveness.


Asunto(s)
Interferón Tipo I/inmunología , Interferón gamma/inmunología , Intestinos/inmunología , Infecciones por Rotavirus/inmunología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Rotavirus
7.
J Virol ; 88(1): 41-53, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24131713

RESUMEN

Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.


Asunto(s)
Interferones/fisiología , Factor de Transcripción STAT1/metabolismo , Proteínas no Estructurales Virales/fisiología , Animales , Células COS , Chlorocebus aethiops , Células HT29 , Humanos , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Interferones/metabolismo , FN-kappa B/antagonistas & inhibidores , Transducción de Señal
8.
PLoS Pathog ; 9(1): e1003064, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23359266

RESUMEN

Viral pathogens must overcome innate antiviral responses to replicate successfully in the host organism. Some of the mechanisms viruses use to interfere with antiviral responses in the infected cell include preventing detection of viral components, perturbing the function of transcription factors that initiate antiviral responses, and inhibiting downstream signal transduction. RNA viruses with small genomes and limited coding space often express multifunctional proteins that modulate several aspects of the normal host response to infection. One such virus, rotavirus, is an important pediatric pathogen that causes severe gastroenteritis, leading to ~450,000 deaths globally each year. In this review, we discuss the nature of the innate antiviral responses triggered by rotavirus infection and the viral mechanisms for inhibiting these responses.


Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Interferones/inmunología , Infecciones por Rotavirus/inmunología , Rotavirus/inmunología , Gastroenteritis/inmunología , Gastroenteritis/virología , Humanos , Inmunidad Innata , Interferones/metabolismo , Rotavirus/crecimiento & desarrollo , Infecciones por Rotavirus/virología , Transducción de Señal/inmunología
9.
Proc Natl Acad Sci U S A ; 109(50): 20667-72, 2012 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-23188796

RESUMEN

"Bulk" measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. Flow cytometry and microfluidics-based single-cell multiplex RT-PCR allowed dissection of IFN responses in single RV-infected and bystander intestinal epithelial cells (IECs). EW replicates in IEC subsets differing in their basal type I IFN transcription and induces IRF3-dependent and IRF3-augmented transcription, but not NF-κB-dependent or type I IFN transcripts. Bystander cells did not display enhanced type I IFN transcription but had elevated levels of certain IFN-stimulated genes, presumably in response to exogenous IFNs secreted from immune cells. Comparison of IRF3 and NF-κB induction in STAT1(-/-) mice revealed that murine but not simian RRV mediated accumulation of IkB-α protein and decreased transcription of NF-κB-dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of "averaged" innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.


Asunto(s)
Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/virología , Animales , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/inmunología , Interferón Tipo I/biosíntesis , Mucosa Intestinal/metabolismo , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Intestino Delgado/virología , Ratones , Ratones de la Cepa 129 , Receptores de Interferón/metabolismo , Rotavirus/inmunología , Rotavirus/patogenicidad , Infecciones por Rotavirus/genética , Infecciones por Rotavirus/metabolismo , Factor de Transcripción STAT1/metabolismo
10.
J Virol ; 87(15): 8307-16, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23698306

RESUMEN

Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.


Asunto(s)
Proteínas de la Cápside/metabolismo , Especificidad del Huésped , Intestinos/virología , Rotavirus/fisiología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Ratones , Ratones Endogámicos BALB C
11.
NPJ Breast Cancer ; 10(1): 40, 2024 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-38839777

RESUMEN

The PI3K, AKT, and mTOR (PAM) pathway is frequently dysregulated in breast cancer (BC) to accommodate high catabolic and anabolic activities driving tumor growth. Current therapeutic options for patients with hormone receptor (HR) + / HER2- advanced BC (ABC) include PAM inhibitors that selectively inhibit only one PAM pathway node, which can lead to drug resistance as cells rapidly adapt to maintain viability. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, may be more effective in BC cells than single-node PAM inhibitors by limiting adaptive resistances. By using multiple functional assays, a panel of BC cell lines was evaluated for their sensitivity to four different PAM inhibitors: gedatolisib (pan-PI3K/mTOR inhibitor), alpelisib (PI3Kα inhibitor), capivasertib (AKT inhibitor), and everolimus (mTORC1 inhibitor). Gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects regardless of the PAM pathway mutational status of the cell lines compared to the single-node PAM inhibitors. The higher efficacy of gedatolisib was confirmed in three-dimensional culture and in BC PDX models. Mechanistically, gedatolisib decreased cell survival, DNA replication, cell migration and invasion, protein synthesis, glucose consumption, lactate production, and oxygen consumption more effectively than the other PAM inhibitors tested. These results indicate that inhibition of multiple PAM pathway nodes by a pan-PI3K/mTOR inhibitor like gedatolisib may be more effective at inducing anti-tumor activity than single-node PAM inhibitors. A global Phase 3 study is currently evaluating gedatolisib plus fulvestrant with and without palbociclib in patients with HR+/HER2- ABC.

12.
J Virol ; 86(19): 10829-40, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22855480

RESUMEN

Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.


Asunto(s)
Inmunoglobulina M/química , Rotavirus/metabolismo , Animales , Linfocitos B/citología , Separación Celular , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo , Humanos , Inmunoglobulina A/química , Inmunoglobulina D/química , Inmunoglobulina G , Inmunoglobulina M/metabolismo , Memoria Inmunológica , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Fenotipo , Infecciones por Rotavirus/metabolismo
13.
J Virol ; 85(8): 3717-32, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21307186

RESUMEN

In mouse embryonic fibroblasts (MEFs), the bovine rotavirus (UK strain) but not the simian rhesus rotavirus (RRV) robustly triggers beta interferon (IFN-ß) secretion, resulting in an IFN-dependent restriction of replication. We now find that both rotavirus strains trigger antiviral transcriptional responses early during infection and that both transcriptional responses and IFN-ß secretion are completely abrogated in MAVS/IPS-1(-/-) MEFs. Replication of UK virus could be rescued in MAVS/IPS-1(-/-) MEFs, and synthesis of viral RNA significantly increased early during virus infection. UK virus induced IFN-ß secretion and transcription of IFN-stimulated genes (ISGs) in both RIG-I(-/-) and MDA-5(-/-) MEFs, and neither receptor was essential by itself for the antiviral response to UK rotavirus. However, when receptors RIG-I and MDA-5 were depleted using RNA interference, we found that both contribute to the magnitude of the IFN response. IRF3 was found to be essential for MAVS/IPS-1-directed ISG transcription and IFN-ß secretion during rotavirus infection. Interestingly, absence of the double-stranded RNA-dependent protein kinase PKR led to a profound defect in the capacity of host cells to secrete IFN-ß in response to virus. Both PKR and IRF3 restricted the early replication of UK as indicated by significant increases in viral RNA in fibroblasts lacking either gene. Despite the loss in IFN-ß secretion in PKR(-/-) MEFs, we did not observe decreased IRF3- or NF-κB-dependent early ISG transcription in these cells. Levels of transcripts encoding IFN-α4, IFN-α5, and IFN-ß were high in infected PKR(-/-) MEFs, indicating that during rotavirus infection, PKR functions at a stage between IFN gene transcription and subsequent IFN-ß secretion. These findings reveal that activation of the antiviral response by rotavirus is dependent on MAVS/IPS-1 and IRF3 and involves both RIG-I and MDA-5 and that IFN-ß secretion during rotavirus infection is regulated by PKR.


Asunto(s)
Regulación de la Expresión Génica , Interferón beta/inmunología , Rotavirus/inmunología , eIF-2 Quinasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Eliminación de Gen , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/biosíntesis , Ratones , Ratones Noqueados , eIF-2 Quinasa/genética
14.
J Virol ; 85(6): 2686-94, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21191030

RESUMEN

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/ß and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1.


Asunto(s)
Proteínas de la Cápside/metabolismo , Vesícula Biliar/virología , Infecciones por Rotavirus/veterinaria , Rotavirus/patogenicidad , Proteínas no Estructurales Virales/metabolismo , Animales , Bovinos , Modelos Animales de Enfermedad , Ratones , Virus Reordenados/genética , Virus Reordenados/patogenicidad , Rotavirus/genética , Infecciones por Rotavirus/virología , Virulencia
15.
Mater Today Proc ; 2022 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-36415746

RESUMEN

Vitamins are the main components of our diet. In our nutrition 14 vitamins are present namely A, B1 (Thiamine), B6(Pyridoxine), B12(Cyanocobalamin), C, D, E, K, niacin, folacin, choline, pantothenic acid and biotin. The main role of it is in treating common diseases like cold. Vitamin C's role in treating pneumonia or Sepsis /Septicemia has been underway for many decades. A great benefit in decreasing the duration of cold is by injecting heavy dose of ascorbic acid. So, at high dose/ risk of injection like it may be obese, diabetes, and the elderly. Vitamin C always acts as an antioxidant that can help to prevent our cells from getting any harm. Recently injection of vitamin C was used in treatment of Covid-19 patients. In this review we have primarily discussed its effects on the immune system and the treatment of pneumonia disorders using vitamin C. At the beginning we have discussed the bio-avalibility of vitamin-C followed by the synthesis of it by plants and animals and then the dietary allowance to be followed for vitamin C regularly. The level of vitamin C is very low in people having pneumonia and those with low immunity are being effected by COVID-19 virus. Kiwi is the main source of vitamin C. Preliminary observational studies show that critically sick individuals use vitamin C for the prevention of the pneumonia to the treatment of the virus COVID-19 by increasing the vitamin C levels in the body.

16.
J Virol ; 83(20): 10322-35, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19656876

RESUMEN

Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.


Asunto(s)
Interacciones Huésped-Patógeno , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Rotavirus/patogenicidad , Proteínas no Estructurales Virales/metabolismo , Animales , Células COS , Bovinos , Línea Celular , Chlorocebus aethiops , Fibroblastos/virología , Regulación Viral de la Expresión Génica , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Ratones , Células 3T3 NIH , Complejo de la Endopetidasa Proteasomal/metabolismo , Rotavirus/clasificación , Rotavirus/fisiología , Especificidad de la Especie , Proteínas no Estructurales Virales/genética
17.
Sci Transl Med ; 9(395)2017 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-28637924

RESUMEN

Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell-sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Rotavirus/inmunología , Adulto , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Antígenos Virales/inmunología , Linfocitos B/inmunología , Proteínas de la Cápside/inmunología , Células Cultivadas , Citometría de Flujo , Humanos , Rotavirus/patogenicidad , Infecciones por Rotavirus/tratamiento farmacológico , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/prevención & control
18.
Virus Res ; 96(1-2): 27-35, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12951263

RESUMEN

Hepatitis C virus (HCV) is a serious human pathogen and an estimated 170 million people are infected worldwide. Current therapeutic regimens have shown limited efficacy against selected genotypes of the virus. The phenomenon of RNA interference can be used to selectively block homologous genes post-transcriptionally, and has revolutionized approaches to study gene function. In this report, we have demonstrated that small interfering RNAs (siRNAs) targeted against NS5A of HCV genotype 1a specifically inhibit NS5A RNA and protein expression in a human hepatoma (HepG2) cell line. Expression of endogenous alpha-actin and the ds-RNA activated serine/threonine kinase-PKR were unaltered, demonstrating that the inhibitory effect observed from siRNA was specific to the HCV NS5A protein. We next examined whether siRNA directed against NS5A could inhibit core protein expression, the first gene product synthesized in virus infected cells due to its localization at the 5' end of the HCV polyprotein. For this purpose, a full-length cDNA clone from HCV (H77, genotype 1a) was used, and results indicated that the introduction of NS5A targeted siRNA resulted in an inhibition of NS5A and core protein expression. Moreover, we observed that this siRNA effectively inhibited NS5A mediated activation of the IL-8 promoter. Taken together, our results demonstrated that siRNA was effective in inhibiting HCV protein expression, and may have therapeutic potential to limit HCV replication in chronically infected patients.


Asunto(s)
Regulación Viral de la Expresión Génica/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Interferencia de ARN/fisiología , ARN Interferente Pequeño/farmacología , Hepacivirus/genética , Hepacivirus/metabolismo , Hepacivirus/fisiología , Humanos , ARN Viral/genética , Células Tumorales Cultivadas , Proteínas no Estructurales Virales , Replicación Viral/efectos de los fármacos
19.
Cell Rep ; 8(2): 633-45, 2014 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-25043183

RESUMEN

Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.


Asunto(s)
Herpesvirus Humano 3/patogenicidad , Tonsila Palatina/citología , Linfocitos T/virología , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Interacciones Huésped-Patógeno , Humanos , Modelos Biológicos , Tonsila Palatina/virología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo
20.
J Virol ; 81(21): 11758-67, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17699573

RESUMEN

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic alpha-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 alpha-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted alpha-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.


Asunto(s)
Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Animales , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Fosforilación , Prolina/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Rotavirus/química , Proteínas no Estructurales Virales/metabolismo
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