Background: The available anti-influenza A drugs show limited efficacy and emergence of drug resistance. The aim of this study is to develop efficient systems for the delivery of small RNA molecules targeting the packaging signal mapping a the 5’ end of the influenza virus genome segments encoding for the viral polymerase (PA, PB1 and PB2) and to assess the efficacy of such nucleic acid based antiviral strategy Methods: We developed different lentiviral vectors expressing microRNAs [pLenti6/V5-GW/EmGFP-miR] or antisense-RNAs [pLL3.7-GFP] targeting the highly conserved sequences of the PA, PB1 and PB2 Influenza A genomic segments. Recombinant lentiviral particles, produced by transfecting human embryonic kidney cells (293T) with both vectors in combination with a "packaging" plasmid systems, were harvested , titrated on human alveolar adenocarcinoma cell line (A549) by FACS analysis, and stored at –80°C. The lentiviral vectors transduction efficiency in different cell lines was evaluated by FACS and quantitative Real-Time PCR. The transduced cells were infected with epidemiologically relevant human type A and B influenza viruses and avian type A viruses. Viral inhibitory activity was analyzed by titrating the viral progeny by an infectivity assay. Results: We showed that recombinant lentiviral particles expressing microRNA or antisense-RNAs efficiently transduced MDCK, A549 and 293T cell lines that are commonly used for influenza virus studies. More importantly a reduction from 1 to 3 logs of the virus titre was obtained in all tested cell lines infected with the different human and avian subtypes of Influenza type A viruses. By contrast, no inhibition of influenza type B virus was observed. Furthermore both microRNAs and antisense-RNAs expressing systems displayed a clear dose-dependent viral inhibitory activity altthough with different efficiency depending on the specific target sequence. Finally, the specificity of the antiviral activity was further assess by infecting the cells with influenza virus clones mutated in the PA, PB1 or PB2 target regions. Conclusions: The expression of small RNAs targeting the packaging signal of the polymerases gene provides protection to the cell host virus infection highlighting their use as a potential candidate for the development of a nucleic acid-based therapeutic strategy against Influenza A virus infection in human and birds.

DEVELOPMENT OF NEW ANTIVIRALS AGAINST THE INFLUENZA TYPE A VIRUSES BASED ON SMALL RNAS TARGETING THE PACKAGING SIGNAL OF THE POLYMERASES GENE CODING REGION

MARTELLI, FRANCESCO;SALATA, CRISTIANO;PAROLIN, MARIA CRISTINA;PALU', GIORGIO;CALISTRI, ARIANNA;
2014

Abstract

Background: The available anti-influenza A drugs show limited efficacy and emergence of drug resistance. The aim of this study is to develop efficient systems for the delivery of small RNA molecules targeting the packaging signal mapping a the 5’ end of the influenza virus genome segments encoding for the viral polymerase (PA, PB1 and PB2) and to assess the efficacy of such nucleic acid based antiviral strategy Methods: We developed different lentiviral vectors expressing microRNAs [pLenti6/V5-GW/EmGFP-miR] or antisense-RNAs [pLL3.7-GFP] targeting the highly conserved sequences of the PA, PB1 and PB2 Influenza A genomic segments. Recombinant lentiviral particles, produced by transfecting human embryonic kidney cells (293T) with both vectors in combination with a "packaging" plasmid systems, were harvested , titrated on human alveolar adenocarcinoma cell line (A549) by FACS analysis, and stored at –80°C. The lentiviral vectors transduction efficiency in different cell lines was evaluated by FACS and quantitative Real-Time PCR. The transduced cells were infected with epidemiologically relevant human type A and B influenza viruses and avian type A viruses. Viral inhibitory activity was analyzed by titrating the viral progeny by an infectivity assay. Results: We showed that recombinant lentiviral particles expressing microRNA or antisense-RNAs efficiently transduced MDCK, A549 and 293T cell lines that are commonly used for influenza virus studies. More importantly a reduction from 1 to 3 logs of the virus titre was obtained in all tested cell lines infected with the different human and avian subtypes of Influenza type A viruses. By contrast, no inhibition of influenza type B virus was observed. Furthermore both microRNAs and antisense-RNAs expressing systems displayed a clear dose-dependent viral inhibitory activity altthough with different efficiency depending on the specific target sequence. Finally, the specificity of the antiviral activity was further assess by infecting the cells with influenza virus clones mutated in the PA, PB1 or PB2 target regions. Conclusions: The expression of small RNAs targeting the packaging signal of the polymerases gene provides protection to the cell host virus infection highlighting their use as a potential candidate for the development of a nucleic acid-based therapeutic strategy against Influenza A virus infection in human and birds.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/2936101
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