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FINLAY ALBARRAN
MEDICAL INSTITUTE
Inhibition of influenza A virus replication by RNA interference targeted against the PB1 subunit of the RNA polymerase gene
Li W, Yang X, Jiang Y, Wang B, Yang Y, Jiang Z, Li M.
Source: Arch Virol. 2011 Aug 12.
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, 610041, Sichuan, China.
Abstract
Influenza (flu) pandemics have posed a great threat to human health in the last century. However, current vaccination strategies and antiviral drugs provide limited protection. RNA interference (RNAi) is an effective means of suppressing influenza virus replication. PB1 is the critical protein subunit of the influenza virus RNA polymerase.
The gene encoding this protein, PB1, is highly conserved among different subtypes of IAV and was therefore chosen as the target in this study. The oligonucleotide, PB1-shRNA, contains a 21-bp siRNA corresponding to nucleotides 1,632 to 1,652 of PB1 linear vRNA with BamHI or EcoRI restriction enzyme sites incorporated at the ends. The PB1-shRNA oligonucleotide was directionally cloned into the RNAi-ready pSIREN-shuttle vector. The correct structure of the resulting pSIREN/PB1 plasmid was confirmed by restriction endonuclease digestion. Madin-Darby canine kidney (MDCK) cells were transfected with pSIREN/PB1 and subsequently infected with IAV at an MOI of 0.1 (A/PR/8/34, H1N1). The virus titer in cell culture supernatants was determined 48 hours later, and it was found that virus growth was inhibited by more than 50-fold relative to controls. Furthermore, embryonated eggs and mice were inoculated with liposome-encapsulated pSIREN/PB1 and then challenged with the A/PR/8/34 virus. The results showed at least a 100-fold inhibition in virus replication in egg allantoic fluid and a survival rate of between 50% and 100% in experimental mice. This study demonstrates that PB1-shRNA expressed by the recombinant plasmid pSIREN/PB1 inhibits influenza A virus replication both in vitro and in vivo. These observations provide a foundation for the development of a new and efficient treatment of influenza infections.
La inhibición de la replicación del virus de la gripe A por la interferencia del ARN dirigida contra la subunidad PB1 del gen de la ARN polimerasa.
Li W, Yang X, Y Jiang, Wang B, Y Yang, Z Jiang, Li M.
Arch Virol. 2011 Ago 12.
Departmento de Microbiología, Universidad de Sichuan, Chengdu, 610041, Sichuan, China.
Las pandemias de influenza (gripe) han planteado una gran amenaza para la salud humana en el siglo pasado y en nuestros tiempos. Sin embargo, las actuales estrategias de vacunación y los medicamentos antivirales proporcionan una protección limitada. ARN de interferencia (ARNi) es un medio eficaz de supresión de la replicación del virus de la influenza. PB1 es la subunidad de la proteína esencial del virus de la influenza de la polimerasa de ARN. El gen que codifica esta proteína, PB1, es altamente conservada entre los diferentes subtipos de IAV y por lo tanto, fue elegido como el objetivo de este estudio. El oligonucleótido, PB1-shRNA, contiene un siRNA de 21 pb correspondiente a los nucleótidos 1632 a 1652 de PB1 lineal ARNv con BamHI y EcoRI sitios de enzima de restricción incorporada en los extremos. El oligonucleótido PB1-ARNhc se clonó direccionalmente en el RNAi listo pSIREN-vector lanzadera. La estructura correcta de la pSIREN/PB1 plásmido resultante fue confirmada por digestión con endonucleasas de restricción. Madin-Darby de riñón canino (MDCK) las células fueron transfectadas con pSIREN/PB1 y posteriormente infectados con IAV en una MOI de 0,1
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