BY:

LAURA VALENTINA CARDONA MONTOYA

MARTÍN ALONSO BARREIRO GARCÍA

MOLECULAR BIOLOGY

2017-1

INTRODUCTION

Japanese encephalitis virus (JEV) is the leading cause of epidemic viral encephalitis worldwide, mainly in East and Southeast Asia. JEV is transmitted by mosquitoes and invades the central nervous system, resulting in the destruction of neuron cells.

JEV is a Falvivirus in the family of the Flaviviridae, is a positive-stranded RNA. The virus encondes a single Methyltransferase which is located in the nonstructural protein NS5. This enzyme is essencial for viral replication and specifically catalayzes methylation of the viral RNA cap. The cap structure is critical for viral genome RNA stability, evasion from host immune response and efficient translation.

JEV MTase might be a potential approach to develop anti-JEV therapy. In this study they identified RNA aptamer against JEV Mtase, it’s a single stranded nucleic acids that can bind to some specific sequences. It could supress JEV replication and production in cell.

JAPANESE ENCEPHALITIS VIRUS

(JEV)

FLAVIVIRIDAE TRANSMITED BY MOSQUITOES

IT DESTROYES NEURON CELLS

VIRUS METHYLTRANSFERASE

IN NS5 PROTEIN

METHYLATION OF VIRAL RNA CAP

RNA STABILITY, TRANSCRIPTION AND INMUNE PROTECTION

INHIBITION OF JEVS MTASE

WILL BE USEFUL

DEVELOP A ANTI-JEV THERAPY

GENERAL OBJECTIVE

Explain the mechanism that was used to create the new Mtase specific RNA aptamer which make an inhibition of Japanse encephalitis virus (JEV) replication.

MATERIALES Y MÉTODOS

VIRUS Y CÉLULAS: JEV y BHK-21 celulas (hamster) fueron donados por Korea Centers for Disease Control and Prevetion.

PROTEINAS RECOMBINANTES: JEV Mtasa, replicasa, y NS5 fueron obtenidas por PCR. Esta técnica se basa en procesos de: desnaturalización, hibridación (primer) y replicación. El objetivo fue obtener múltiples copias o clones del RNA del virus.

Gen: 5’-CGGGATCCGCCTTAGAAGGCCTGGGGGCAG-3’

RT PCR: se utilizó para la amplificación del ARN a partir de la síntesis previa de su DNAc (copia), utilizaron una transcriptasa inversa y varios ciclos de PCR convencional, con esta se pudo determinar la expresión del gen de la MTasa.

Northern blot: permitió identificar secuencias especificas de RNA, no se utilizan enzimas de restricción pues son moléculas muy pequeñas. Se aisló el RNA y se realizó electroforesis en gel de agarosa.

PCRq: Se introdujo Bromuro de Etidio para que se uniera a los ácidos nucleicos. Este compuesto emite la fluorescencia proporcional a la cantidad de ácidos nucleicos, de esta manera, permitió cuantificar la cantidad de RNA.

Recombinantes: se utilizó como vector el plásmido pJFH-1.

Inmunoblot: Después de la electroforesis, se transfiere el RNA a un filtro, se incuba con anticuerpos específicos que reaccionan con las proteínas de interés. Así, se identifica la proteina contra la cual está dirigido el anticuerpo. Anticuerpos: Monoclonal de ratón anti-m7G cap y IgG-HRP de cabra anti-ratón. Se visualizó en película de rayos X.

RESULTS

FIG. 1

In this electrophoresis we use tRNA and Library RNA as controls; these two components should show no bind to MTase. We use a G2 aptamer and G2 mutant to check the effectiveness of the G2 aptamer and to check which part of the G2 aptamer makes the inhibition (G2 mutant) the G2 aptamer was effective and G2 mutant was not.

In this electrophoresis we check the G2 aptamer and G2 mutant bind to JEV different components, G2 aptamer only bind to MTase and NS5, G2 mutant did not show any bind. They used EtBr.

FIG 2

In this electrophoresis we confirm that the purified recombinant JEV MTase only methylate the JEV 5’ UTR. We use m7G RNA to check that methylation. Here we confirm the G2 aptamer inhibition of MTase methylation. We use a m7G RNA to probe it. RNA only and negative MTase were use as controls. MTase positive and G2 mutant were positive to methylation. RNA only, negative MTase and G2 aptamer were negative to methylation.

FIG 3

In this we can see which part of the G2 aptamer have the ability to bind to MTase. Right part was the one that could bind. The inputs were used as controls. After they took the only part with the ability to bind to MTase, they modificated to study if it was still able to bind. 24 mer mutant lost that ability. The input was used as control.

In this electrophoresis we can see again that 24 mer aptamer makes an inhibition on the MTase, also that 24 mer mutant have no effect on MTase.

DISCUSSION

CONCLUSIONS

Through the recognition of important enzymes in the replication of virus we can created several treatments against them, in this case the recognition of the Mtase and the search for an inhibitor is an excellent example.

Using several methods like electrophoresis, allowed to check in the article the function of the aptamers, choose which one was the more effective and which part of the aptamer had the effect on the inhibited enzyme.

The use of therapies that have this specificity of only work in the virus proliferation protect our cells of the damage that other non-specific component creating a bigger therapeutic range.

The method used in the JEV Mtase was completely effective and should be use in other virus that have the similarities with these one or that have specific enzymes important in their replication.

Por: Laura

By: Martín

BIBLIOGRAFÍA:

Department of Integrated Life Sciences, Research Institute of Advanced Omics, Dankook University, Yongin, Republic of Korea. (2016, Diciembre 13) Inhibition of Japanese encephalitis virus (JEV) replication by specific. Science Direct. http://www.sciencedirect.com/science/article/pii/S0006291X16321258

MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 8va ed. Medellín: UPB. Fac. de Medicina, 2017. 160, 182 p.