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
Author
Rodríguez Rodríguez, PalomaEntity
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Date
2016-07-07Funded by
Beca predoctoral de la Junta para la Ampliación de estudios (JAE) del Consejo Superior de Investigaciones Científicas.Subjects
Virus de la gripe - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 07-07-2016Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
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.
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