Mutational Analysis of the Different Bulge Regions of Hepatitis C Virus Domain II and Their Influence on Internal Ribosome Entry Site Translational Ability

The hepatitis C virus (HCV) 5′-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation

A search for structurally similar cellular internal ribosome entry sites

Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function

HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

The HCV internal ribosome entry site (IRES) spans a region of ∼340 nt that encompasses most of the 5′ untranslated region (5′UTR) of the viral mRNA and the first 24–40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5′UTR on IRES activity, naturally occurring variants of the 5′UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation

Información Investigador: Odreman Macchioli, Rodolfo Oscar

Resumen Curricular Médico cirujano egresado de la Universidad de Los Andes. Especialista en Cardiología de la Universidad de los Andes (1995). Fellowship en Ecocardiografía de Adultos en el Hospital Universitario Gredorio Marañón de Madrid, España, dependiente de la Universidad Complutense de Madrid. Actualmente coordinador del Laboratorio de Ecocardiografía del Instituto de Investigaciones Cardiovasculares de la Universidad de Los Andes. Actualmente adscrito al Programa de Promoción al Investigador (PPI) como Candidato bajo el número PPI 05921.Post./Espec.5921Candidato - 200355 - 2005; 12 - 2003Ecocardiografía. Ecocardiografía de estrés. Sistema Neurohormonal e Insuficiencia cardiaca. Miocardiopatía chagásica.Marzo de 2007Médico+58 274 2403230Facultad de [email protected], [email protected]

Evaluación de la frecuencia cardíaca y su relación con el remodelado ventricular en respuesta al ejercicio dinámico como indicador de función autonómica en atletas, pesistas y maratonistas

This study was performed to evaluate 118 subjects (athlete’s runners, weight lifters, with control group of healthy subjects sedentary) who have performed stress with treadmill test to evaluate the vagal reserve, as the grade of acceleration of the heart rate (HR) after the 10 second of starting the exercise and the Δ1 and Δ2 respectively,. Transtoracic echocardiography was performed to evaluate diameters and ventricular volumes and diastolic function to determine the patterns of ventricular filling. The sample was divided into three groups: Group I Sedentary, Group II Runners, and the Group III Weight Lifters. Group II was those with vagal predominance in the test with a Δ1 de m=36±14 lat/min and Δ2 de m=58±15 lat/min (p0.0001 and expressed left ventricular (LV) geometric patterns given by higher LV diameter and higher volumes indexed (m= 63±82 mm) p 0.0001 in relation to the group I and III. On the other hand, the weight lifters expressed the less HR recovery after the exercise and the higher wall thickness and higher relation E/e (m=8±3). p0.0001. This study allows us to affirm on the vagal predominance and less stiffness, was observed in endurance athletes, compared to athlete’s static and also allows opening a light to the explanation of the possible reasons to elucidate this phenomenon.Se estudiaron 113 sujetos (atletas maratonistas, atletas pesistas y controles sedentarios sanos) a los que se les realizó una prueba de esfuerzo en banda sin fin, para evaluar la reserva vagal, definida por la aceleración de la frecuencia cardiaca (FC) a los 10 segundos de iniciado el ejercicio, como la recuperación de la FC al primer minuto y el segundo minuto después de la realización del ejercicio dinámico, Δ1 y Δ2 respectivamente. Se realizó ecocardiograma transtoraxico para evaluar los índices de volúmenes ventriculares y la función diastólica, para determinar los patrones de llenado ventricular. La muestra se dividió en tres grupos: Grupo I: Control de Sedentarios Sanos, Grupo II: Maratonistas, Grupo III: Pesistas. Se observó que el Grupo II mostro mayor recuperación de la FC con un Δ1 de m=36±14 lat/min y Δ2 de m=58±15 lat/min, con mayor predominio vagal; asociado con un patrón geométrico ventricular izquierdo (VI) de mayores volúmenes indexados del VI de m= 63±82 mm con una p 0,0001 con relación a los del Grupo I y Grupo III. El Grupo III mostró menor recuperación de la FC con respecto a los demás grupos y mayor índice de la relación E/e’ (m=8±3), p 0,0001. Por lo tanto, este estudio nos permite concluir que los atletas mostraron un tono vagal aumentado y una menor rigidez VI con respecto a atletas estáticos y plantea los posibles mecanismos que explican este fenómeno

Dinamic changes of pro-inflammatory cytokines, adhesion molecules and lymphocytes activation markers as early indicators of diseases severity in patients with Dengue

Several immunopathogenic mechanisms have been proposed to explain the massive increase of vascular permeability observed in the severe forms of infection by Dengue Virus (DENV). Our aim was to determine the kinetic changes of inflammatory mediators (IL-8, TNF- α), soluble early lymphocyte activation markers (sIL-2R, sTNF-Rp75) and soluble fractions of cell adhesion molecules (sICAM-1 and sVCAM-1) as indicators for early recognition of disease severity in patients with laboratory-confirmed dengue. Twenty patients classified as Dengue±Warning Signs (D±WS) and thirty patients with Severe Dengue (SD) were included in the study. Serums of apparently healthy individuals were included as controls. Compared with normal subjects, D±WS cases did not show significant differences in the levels of IL-8 or TNF-α during the acute nor in the critical stages of the disease; however, in D±WS cases levels of sICAM-1 and sVCAM-1 were higher than controls during both phases; in contrast, significant increase of sTNF-p75 and sIL2R levels were observed during the critical phase of the disease. Compared with both dengue patients and controls, patients with SD showed significant rise in the levels of IL-8 and TNF-α during the critical phase of the disease and a significant increase in adhesion molecules were detected in both phases, but the highest levels of sVCAM-1 and sIL-2R were observed only during the acute stage of the disease. In conclusion, sIL-2R and sVCAM-1, as early markers of lymphocyte and endothelial activation, would serves as indicators of severity during the acute phase of dengue infection