title: Packaging signals in the 5'-ends of influenza virus PA, PB1, and PB2 genes as potential targets to develop nucleic-acid based antiviral molecules.
abstract: In a previous study a 15-mer phosphorothioate oligonucleotide (S-ON) derived from the packaging signal in the 5' end of segment 1 (PB2) of influenza A virus (designated 5-15b) proved markedly inhibitory to virus replication. Here we investigated whether analogous inhibitory S-ONs targeting the 5' end of segments 2 (PB1) and 3 (PA) could be identified and whether viral resistance to S-ONs can be developed. Similar to our earlier result, 20-mer S-ONs reproducing the 5' ends of segments 2 or 3 (complementary to the 3'-coding regions of PB1 and PA, respectively) exerted a powerful antiviral activity against a variety of influenza A virus subtypes in MDCK cells. Serial passage of the A/Taiwan/1/86 H1N1 strain in the presence of S-ON 5-15b or its antisense as5-15b analogue showed that mutant viruses with reduced susceptibility to the S-ON could indeed be generated, although the resistant viruses displayed reduced replicative fitness. Sequencing the resistant viruses identified mutations in the PB1, PB2, PA and M1 genes. Introduction of these changes into the A/PR/8/34 H1N1 strain by reverse genetics, suggested that alterations to RNA function in the packaging regions of segments 2 and 3 were important in developing resistance to S-ON inhibition. However, many of the other sequence changes induced by S-ON treatment were markedly deleterious to virus fitness. We conclude that packaging signals in the influenza A virus polymerase segments provide feasible targets for nucleic acid-based antivirals that may be difficult for the virus to evade through resistance mutations.
authors: Virology Unit, Department of Public Health, University of Florence, Florence, Italy. simone.giannecchini@unifi.it
authors: Simone
authors: S
authors: Giannecchini
authors: Helen M
authors: HM
authors: Wise
authors: Paul
authors: P
authors: Digard
authors: Valeria
authors: V
authors: Clausi
authors: Edoardo
authors: E
authors: Del Poggetto
authors: Liberio
authors: L
authors: Vesco
authors: Simona
authors: S
authors: Puzelli
authors: Isabella
authors: I
authors: Donatelli
authors: Alberta
authors: A
authors: Azzi
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D024921:Drug Resistance, Multiple, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D009980:Influenza A virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D054735:Phosphorothioate Oligonucleotides / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; C061290:PA protein, influenza viruses; C502893:PB2 protein, influenza virus; D054735:Phosphorothioate Oligonucleotides; D012367:RNA, Viral; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase