Nueva estrategia basada en la utilización de aptámeros como antivirales contra el virus de la gripe y estudio de los mecanismos de control de la expresión génica viral

Resumen

The influenza A viruses (IAV) are members of the Orthomyxoviridae family & causes respiratory disease & continue to be one of the largest global threats to human health, causing 250.000-500.000 deaths per year & these numbers can be much larger in a pandemic. The virus possesses a negative-oriented segmented RNA genome that encodes for its own RNA-dependent RNA polymerase, which is error-prone. In addition, its segmented genome allows for exchange of RNA segments between genotypically different influenza viruses. These features confer a high genetic diversity & lead to generation of novel strains or/& subtypes & thus contribute to the permanent exposure of the human population to newly emerging & re-emerging influenza viruses. Although at present we have effective influenza antivirals that reduce virus dissemination, the appearance of resistant mutant viruses poses a serious limitation for their widespread use. Antivirals drugs that target cellular proteins may play major roles in combating virus resistance. Influenza virus mRNAs are formally equivalent to the cellular mRNAs & a sophisticated strategy has been selected by the virus to enhance specifically the translation of viral mRNAs. In this context, previous work has demonstrated the role of NS1 & viral polymerase establishing productive interactions with eIF4GI translation initiation factor & with the polyA binding protein 1 (hPABP1). Thus, the inhibition of viral proteins-translation factors interactions or their destabilization can be potentialy used as an antiviral strategy. Recently, nucleic acid aptamers have been put forward for use as therapeutic agents against many human diseases due to their inhibitory ability & target specificity. In the present study, we have selected ssDNA aptamers specific to the human hPABP1, as possible inhibitors of IAV mRNA translation & as potential antivirals for influenza virus replication. Two aptamers (ApPABP7 & ApPABP11), which bind hPABP1 with high affinity have been characterized. Both aptamers inhibit influenza virus replication of H1N1 or H3N2 subtypes at high & low multiplicity of infection & the viral polymerase-eIF4GI interaction. In addition, aptamer ApPABP11 inhibits the interactions between NS1 & eIF4GI or hPABP1. These results provide support for a potential use of aptamers targeting viral-cellular interactions as novel antivirals against influenza virus replication. In order to analyze the specific contribution of segment-specific sequences located adjacently to the promoter conserved nucleotides, we have generated a mutant library at the four adjacent nucleotides in the NS segment. The different mutants have been examined in a RNP reconstitution assay as well as in recombinant mutant viruses. A thorough analysis of these mutants shows that none of the mutations affect viral polymerase activity, while recombinant viruses that contain mutations in the first nucleotide adjacent to the viral promoter, producea marked decreased accumulation of NS1. In addition, these mutants decrease viral proteins accumulation mainly at the early stages of the infection & nuclear retention of viral RNPS. Consequently these mutant viruses have a delayed production of infective particles with lower viral titres at one & multiple step curves.