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2.
Am J Gastroenterol ; 118(1): 138-147, 2023 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-36113491

RESUMEN

INTRODUCTION: Patients with ulcerative colitis (UC) regard rapid onset of action among the most important aspects of their treatment. We used the partial Mayo Clinic Score (pMCS) and component patient-reported subscores to assess the rapidity and sustainability of response to filgotinib, a once-daily, oral Janus kinase 1 preferential inhibitor, in adults with moderately to severely active UC in the phase 2b/3 SELECTION trial. The association between early symptomatic improvements and health-related quality of life (HRQoL) outcomes was also assessed. METHODS: In these post hoc analyses of the double-blinded, randomized, placebo-controlled 58-week SELECTION trial (NCT02914522), rectal bleeding and stool frequency diary data on days 1-15 and pMCS remission and response at multiple time points including weeks 10 and 58 were evaluated. HRQoL was assessed using the Inflammatory Bowel Disease Questionnaire at weeks 10 and 58. RESULTS: Filgotinib 200 mg relative to placebo improved rectal bleeding and stool frequency within 7 days ( P < 0.05). By week 2, greater proportions of filgotinib 200 mg-treated patients than placebo-treated patients achieved pMCS remission (biologic-naive, 15.1% vs 8.0%, P = 0.0410; biologic-experienced, 10.3% vs 4.2%, P = 0.0274). A similar treatment effect was observed at week 58 ( P < 0.0001). Day 7 rectal bleeding and stool frequency subscores were associated with the Mayo Clinic Score response at weeks 10 and 58. Patients in pMCS remission at weeks 10 and 58 had greater improvements in the Inflammatory Bowel Disease Questionnaire score than those not in pMCS remission. DISCUSSION: Filgotinib 200 mg daily resulted in rapid and sustained improvements in both UC symptoms and HRQoL.


Asunto(s)
Productos Biológicos , Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Inhibidores de las Cinasas Janus , Adulto , Humanos , Colitis Ulcerosa/tratamiento farmacológico , Calidad de Vida , Inhibidores de las Cinasas Janus/uso terapéutico , Productos Biológicos/uso terapéutico , Método Doble Ciego , Inducción de Remisión , Resultado del Tratamiento
3.
Open Forum Infect Dis ; 6(4): ofz090, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31024970

RESUMEN

BACKGROUND: Staphylococcus aureus (SA) bacteremia often requires a long treatment duration with antibiotics to prevent relapse due to the ability of SA to establish reservoirs of infection in sites such as heart and bone. These metastatic sites of infection cannot be serially sampled to monitor the clearance of SA infection. This study aimed to establish a link between persistence of circulating SA deoxyribonucleic acid (SA-DNA) and tissue reservoirs in patients with SA bacteremia. METHODS: A highly sensitive quantitative polymerase chain reaction was used to measure whole blood SA-DNA and plasma-derived SA cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 73 patients with confirmed SA bacteremia and correlated with clinical features. RESULTS: Blood SA-DNA was detected for longer than the duration of positive blood cultures. Longer duration of circulating bacterial DNA was observed in complicated SA bacteremia infections, such as endocarditis and osteoarticular infections, compared with uncomplicated bloodstream infections. In contrast, traditional blood cultures demonstrated similar time to clearance regardless of foci of infection. Plasma-derived SA-cfDNA showed concordance with blood SA-DNA levels. Baseline levels of SA-DNA were higher in patients presenting with greater clinical severity and complicated bacteremia. CONCLUSIONS: Prolonged levels of circulating SA-DNA in patients with complicated tissue reservoirs after clearance of blood cultures observed in this single-center study should be validated in additional cohorts to assess the potential utility for monitoring clearance of infection in patients with SA bacteremia.

4.
Clin Infect Dis ; 68(9): 1502-1511, 2019 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-30165412

RESUMEN

BACKGROUND: Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies. METHODS: To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124). RESULTS: We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012). CONCLUSIONS: These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.


Asunto(s)
Bacteriemia/diagnóstico , Quimiocina CCL2/sangre , Endocarditis Bacteriana/diagnóstico , Interleucina-8/sangre , Infecciones Estafilocócicas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Bacteriemia/complicaciones , Bacteriemia/tratamiento farmacológico , Bacteriemia/mortalidad , Biomarcadores/sangre , Estudios de Casos y Controles , Endocarditis Bacteriana/complicaciones , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/mortalidad , Femenino , Humanos , Interleucina-17/sangre , Masculino , Persona de Mediana Edad , Pronóstico , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/patogenicidad , Análisis de Supervivencia
5.
EBioMedicine ; 13: 321-327, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27742226

RESUMEN

BACKGROUND: Initiation of antiretroviral therapy (ART) and subsequent virologic suppression reduces immune activation and systemic inflammation. METHODS: We examined longitudinal changes in biomarkers of monocyte activation (sCD14, sCD163), and systemic (IL-6, hsCRP, sTNFR-I and D-dimer) and vascular (Lp-PLA2) inflammation in a subgroup (N=100 per arm) of participants enrolled in a randomized, placebo-controlled trial comparing elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF; TAF) to E/C/F/tenofovir disoproxil fumarate (E/C/F/TDF; TDF) in treatment-naïve adults. RESULTS: For 194 participants (TAF, 98; TDF, 96), baseline levels of biomarkers did not differ by treatment arm; there were no differences in biomarker values between groups at weeks 12, 24, or 48 (p>0.05), except IL-6 at week 12 (p=0.012). Among all participants (combining groups), there were statistically significant declines from baseline observed for D-dimer, sCD163, and sTNFR-1 by week 12 and IL-6 by week 24. The proportion of participants with Lp-LA2 levels<200ng per mL (p=0.250) or hsCRP levels <3000mg per L (p=0.586) was unchanged through week 48. CONCLUSIONS: We observed equivalent declines in biomarkers of monocyte activation and systemic inflammation in treatment-naïve adults treated with TAF or TDF for 48weeks, suggesting that TAF and TDF have equivalent impact on immune activation and inflammation.


Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/sangre , Infecciones por VIH/virología , VIH-1 , Mediadores de Inflamación/sangre , Tenofovir/uso terapéutico , Adenina/farmacología , Adenina/uso terapéutico , Adulto , Alanina , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa , Biomarcadores , Comorbilidad , Femenino , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Masculino , Curva ROC , Tenofovir/farmacología
6.
MAbs ; 8(8): 1612-1619, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27653831

RESUMEN

DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK.


Asunto(s)
Antibacterianos/farmacocinética , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacocinética , Inmunoconjugados/farmacocinética , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Ratones , Staphylococcus aureus
7.
J Virol ; 85(17): 9167-75, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21715484

RESUMEN

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8(+) T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8(+) T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4(+) T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8(+) T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.


Asunto(s)
Antirretrovirales/administración & dosificación , Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/genética , Mutación Missense , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Sustitución de Aminoácidos/genética , Animales , Linfocitos T CD8-positivos/inmunología , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Epítopos Inmunodominantes/inmunología , Macaca , Selección Genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología
8.
J Clin Virol ; 51(3): 195-8, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21550842

RESUMEN

The mechanism of elite control of HIV-1 replication is not fully understood. While immunosuppression due to rituximab based chemotherapy has been associated with increased replication of HBV, CMV, and HIV-1, control of replication-competent HIV-1 was maintained in an elite controller/suppressor treated with a regimen that included vincristine, cyclophosphamide, prednisone, four rounds of plasmapheresis and ten cycles of rituximab. The data suggests that de-novo antibody responses do not play a significant role in the control of viral replication in these patients.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Inmunosupresores/administración & dosificación , Quimioterapia/métodos , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Rituximab , Análisis de Secuencia de ADN
9.
AIDS Res Hum Retroviruses ; 26(12): 1307-11, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20854198

RESUMEN

The HLA-B*27 allele is overrepresented in patients who control HIV-1 replication without antiretroviral therapy. CD8(+) T cell responses that target the immunodominant KK10 epitope in Gag are thought to play a major role in this control, and escape at R264 of KK10 is often associated with dramatic virologic breakthrough. We present a case in which an HLA-B*27-positive chronic progressor transmitted HIV-1 to an HLA-B*27-positive viremic controller who was temporarily on HAART, but who has since controlled viremia for over 4 years. We hypothesized that differences in the KK10 epitope of these patients would affect pathogenesis and viral fitness, but found no correlation between autologous KK10 mutations and disease progression or between the predicted fitness impact of autologous HLA-B*27-associated mutations and the actual fitness of autologous virus. This case of viral transmission between two HLA-B*27-positive individuals provides further evidence that prolonged control of fully pathogenic HIV-1 is possible.


Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Sobrevivientes de VIH a Largo Plazo , VIH-1/inmunología , VIH-1/aislamiento & purificación , Antígeno HLA-B27/genética , Epítopos de Linfocito T/genética , Infecciones por VIH/tratamiento farmacológico , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Carga Viral , Privación de Tratamiento , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética
11.
Clin Infect Dis ; 49(11): 1763-6, 2009 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-19857162

RESUMEN

Elite controllers or suppressors are untreated human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain undetectable viral loads. In this study, we show that most elite suppressors do not experience significant changes in T cell counts over a 10-year period. Interestingly, treatment of an elite suppressor with highly active antiretroviral therapy (HAART) led to a marked decrease in immune activation.


Asunto(s)
Antivirales/farmacología , Antivirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Linfocitos T/efectos de los fármacos , Adulto , Terapia Antirretroviral Altamente Activa/efectos adversos , Antivirales/efectos adversos , Recuento de Linfocito CD4 , Estudios de Cohortes , Humanos , Recuento de Linfocitos , Persona de Mediana Edad , Linfocitos T/inmunología , Carga Viral/efectos de los fármacos
12.
J Clin Invest ; 119(11): 3473-86, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19805909

RESUMEN

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-kappaB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.


Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Activación de Linfocitos/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Transducción Genética
13.
J Virol ; 83(18): 9247-57, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19570871

RESUMEN

The treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART), a combination of three or more antiretroviral drugs, suppresses viremia below the clinical limit of detection (50 HIV-1 RNA copies/ml), but latently infected resting CD4(+) T cells serve as lifelong reservoirs, and low-level viremia can be detected with special assays. Recent studies have provided evidence for additional reservoirs that contribute to residual viremia but are not present in circulating cells. Identification of all the sources of residual viremia in humans may be difficult. These discoveries highlight the need for a tractable model system to identify additional viral reservoirs that could represent barriers to eradication. In this study, simian immunodeficiency virus (SIV)-infected pig-tailed macaques (Macaca nemestrina) were treated with four antiretroviral drugs to develop an animal model for viral suppression during effective HAART. Treatment led to a biphasic decay in viremia and a significant rise in levels of circulating CD4(+) T cells. At terminal infection time points, the frequency of circulating resting CD4(+) T cells harboring replication-competent virus was reduced to a low steady-state level similar to that observed for HIV-infected patients on HAART. The frequencies of resting CD4(+) T cells harboring replication-competent virus in the pooled head lymph nodes, gut lymph nodes, spleen, and peripheral blood were reduced relative to those for untreated SIV-infected animals. These observations closely parallel findings for HIV-infected humans on suppressive HAART and demonstrate the value of this animal model to identify and characterize viral reservoirs persisting in the setting of suppressive antiretroviral drugs.


Asunto(s)
Terapia Antirretroviral Altamente Activa/métodos , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus , Animales , Linfocitos T CD4-Positivos/virología , Modelos Animales de Enfermedad , Ganglios Linfáticos/virología , Recuento de Linfocitos , Macaca , Viremia/tratamiento farmacológico
14.
Clin Infect Dis ; 47(1): 102-4, 2008 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-18494606

RESUMEN

Elite suppressors are untreated individuals with human immunodeficiency virus type 1 (HIV-1) infection who maintain viral loads <50 copies/mL. Using a single-copy assay, we show that there is no statistically significant difference between the proportions of elite suppressors and patients receiving suppressive highly active antiretroviral therapy who have viral loads of <1 copy/mL.


Asunto(s)
Terapia Antirretroviral Altamente Activa , Infecciones por VIH/virología , Sobrevivientes de VIH a Largo Plazo , VIH-1/aislamiento & purificación , Carga Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos
15.
Proc Natl Acad Sci U S A ; 105(12): 4832-7, 2008 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-18362342

RESUMEN

The time to suppression of HIV-1 viremia to below the limit of detection of standard clinical assays is an important prognostic indicator for patients on highly active antiretroviral therapy (HAART). Recent clinical trials of the integrase inhibitor raltegravir have demonstrated more rapid viral decay than previously seen with reverse transcriptase (RT) or protease inhibitor-based regimens. Because of the therapeutic importance of drugs that target different steps in the virus life cycle, it is imperative to consider whether viral dynamics are affected by the stage of the viral life cycle at which an antiretroviral drug acts. We use a mathematical model to investigate the effects of various drug classes on the dynamics of HIV-1 decay and show that the stage at which a drug acts affects the dynamics of viral decay. We find that the drug class acting latest in the viral life cycle dictates the dynamics of HIV-1 decay. In general, we find that the later in the life cycle an inhibitor acts, the more rapid the decay in viremia, and we illustrate this by comparing the effect of RT and integrase inhibitors on viral dynamics. We conclude that the rapid decay observed in patients on integrase-inhibitor-containing regimens is not necessarily an indication of greater drug efficacy but rather an expected consequence of the fact that this drug acts later in the life cycle. We propose that clinically observed viral decay rates for HAART regimens should be evaluated in the context of the drug classes that are represented.


Asunto(s)
VIH-1/fisiología , Terapia Antirretroviral Altamente Activa , Antivirales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Inhibidores de Integrasa/farmacología , Modelos Biológicos , Inhibidores de la Transcriptasa Inversa/farmacología , Viremia/virología
16.
Mol Cell Proteomics ; 6(6): 1027-38, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17339631

RESUMEN

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein muNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase lambda3, that was recruited to the structures through an association with muNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of muNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the muNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent muNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.


Asunto(s)
Orthoreovirus/química , Proteínas Virales/metabolismo , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Células COS , Chlorocebus aethiops , Estructuras Citoplasmáticas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo
17.
J Virol ; 78(19): 10291-302, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15367595

RESUMEN

Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the mu2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the mu2-encoding M1 genome segment, although contributions by the lambda3- and lambda2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated mu2 (Ub-mu2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by mu2 from globular strains but not with microtubules coated by mu2 from filamentous strains, and immunoprecipitations revealed that mu2 from globular strains displayed higher levels of Ub-mu2. Allelic changes at mu2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their mu2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that mu2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-mu2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.


Asunto(s)
Cuerpos de Inclusión Viral/ultraestructura , Orthoreovirus/crecimiento & desarrollo , Orthoreovirus/genética , Ubiquitina/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Genes Virales , Cuerpos de Inclusión Viral/metabolismo , Microtúbulos/química , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Orthoreovirus/metabolismo , Fenotipo , Pliegue de Proteína , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Virus Reordenados/genética , Virus Reordenados/crecimiento & desarrollo , Virus Reordenados/metabolismo , Temperatura , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/genética
18.
Biochem Biophys Res Commun ; 317(2): 648-53, 2004 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-15063807

RESUMEN

The Saccharomyces cerevisiae gene ABC1 was originally isolated as a multicopy suppressor of a yeast strain harboring a mutation in a cytochrome b translational activator (cbs2-223). Based on this identification, Abc1p was postulated to activate the bc1 complex and function as a chaperone of cytochrome b. ABC1 was subsequently identified as COQ8 and found to be necessary for yeast coenzyme Q synthesis. In this work we show that a segment of yeast genomic DNA containing ABC1/COQ8 and neighboring genes suppresses the respiratory and Q-deficient phenotypes of the coq6 mutant, coq6-1. COQ6 is essential for yeast coenzyme Q biosynthesis. We show that a tRNA(TRP) gene located downstream of ABC1/COQ8 mediates suppression of the cbs2-223 and coq6-1 mutations, and each is identified here as containing UGA nonsense codons. The inability of ABC1/COQ8 to suppress the cbs2-223 allele in multicopy indicates it may not be a chaperone as previously reported.


Asunto(s)
Regulación Fúngica de la Expresión Génica/fisiología , ARN de Transferencia de Triptófano/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supresión Genética/genética , Transactivadores/metabolismo , Ubiquinona/metabolismo , Chaperonas Moleculares , Mutagénesis Sitio-Dirigida , ARN de Transferencia de Triptófano/genética , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Especificidad de la Especie , Transactivadores/genética , Ubiquinona/genética
19.
J Virol ; 78(4): 1882-92, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14747553

RESUMEN

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein mu NS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with mu NS. We found that all three of these proteins--lambda 1, lambda 2, and sigma 2--localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with mu NS, on the other hand, each core surface protein colocalized with mu NS in globular inclusions, supporting the initial hypothesis. We also found that lambda 1, lambda 2, and sigma 2 each localized to filamentous inclusions formed upon the coexpression of mu NS and mu 2, a structurally minor core protein that associates with microtubules. The first 40 residues of mu NS, which are required for association with mu 2 and the RNA-binding nonstructural protein sigma NS, were not required for association with any of the three core surface proteins. When coexpressed with mu 2 in the absence of mu NS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with mu 2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of mu NS that had been preformed in those cells, providing evidence that mu NS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins mu NS and mu 2 in this process.


Asunto(s)
Cuerpos de Inclusión Viral/metabolismo , Reoviridae/metabolismo , Proteínas del Núcleo Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus , Animales , Proteínas de la Cápside , Línea Celular , Proteínas de Unión al ADN , Humanos , Ratones , Microscopía Fluorescente , Proteínas de Unión al ARN , Proteínas Virales/metabolismo , Virión/metabolismo
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