Secondary structure of the segment 5 genomic RNA of influenza A virus and its application for designing antisense oligonucleotides.

Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (-)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics.

A Short Chemically Modified dsRNA-Binding PNA (dbPNA) Inhibits Influenza Viral Replication by Targeting Viral RNA Panhandle Structure.

RNAs play critical roles in diverse catalytic and regulatory biological processes and are emerging as important disease biomarkers and therapeutic targets. Thus, developing chemical compounds for targeting any desired RNA structures has great potential in biomedical applications. The viral and cellular RNA sequence and structure databases lay the groundwork for developing RNA-binding chemical ligands through the recognition of both RNA sequence and RNA structure. Influenza A virion consists of eight segments of negative-strand viral RNA (vRNA), all of which contain a highly conserved panhandle duplex structure formed between the first 13 nucleotides at the 5' end and the last 12 nucleotides at the 3' end. Here, we report our binding and cell culture anti-influenza assays of a short 10-mer chemically modified double-stranded RNA (dsRNA)-binding peptide nucleic acid (PNA) designed to bind to the panhandle duplex structure through novel major-groove PNA·RNA2 triplex formation. We demonstrated that incorporation of chemically modified PNA residues thio-pseudoisocytosine (L) and guanidine-modified 5-methyl cytosine (Q) previously developed by us facilitates the sequence-specific recognition of Watson-Crick G-C and C-G pairs, respectively, at physiologically relevant conditions. Significantly, the chemically modified dsRNA-binding PNA (dbPNA) shows selective binding to the dsRNA region in panhandle structure over a single-stranded RNA (ssRNA) and a dsDNA containing the same sequence. The panhandle structure is not accessible to traditional antisense DNA or RNA with a similar length. Conjugation of the dbPNA with an aminosugar neamine enhances the cellular uptake. We observed that 2-5 µM dbPNA-neamine conjugate results in a significant reduction of viral replication. In addition, the 10-mer dbPNA inhibits innate immune receptor RIG-I binding to panhandle structure and thus RIG-I ATPase activity. These findings would provide the foundation for developing novel dbPNAs for the detection of influenza viral RNAs and therapeutics with optimal antiviral and immunomodulatory activities.

Influenza virus segment 5 (+)RNA - secondary structure and new targets for antiviral strategies.

Influenza A virus is a threat for humans due to seasonal epidemics and occasional pandemics. This virus can generate new strains that are dangerous through nucleotide/amino acid changes or through segmental recombination of the viral RNA genome. It is important to gain wider knowledge about influenza virus RNA to create new strategies for drugs that will inhibit its spread. Here, we present the experimentally determined secondary structure of the influenza segment 5 (+)RNA. Two RNAs were studied: the full-length segment 5 (+)RNA and a shorter construct containing only the coding region. Chemical mapping data combined with thermodynamic energy minimization were used in secondary structure prediction. Sequence/structure analysis showed that the determined secondary structure of segment 5 (+)RNA is mostly conserved between influenza virus type A strains. Microarray mapping and RNase H cleavage identified accessible sites for oligonucleotides in the revealed secondary structure of segment 5 (+)RNA. Antisense oligonucleotides were designed based on the secondary structure model and tested against influenza virus in cell culture. Inhibition of influenza virus proliferation was noticed, identifying good targets for antisense strategies. Effective target sites fall within two domains, which are conserved in sequence/structure indicating their importance to the virus.

Self-Folding of Naked Segment 8 Genomic RNA of Influenza A Virus.

Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by in vitro chemical structure mapping, thermodynamics, bioinformatics, and binding to isoenergetic microarrays of an entire natural sequence of the 875 nt segment 8 vRNA and of a smaller fragment. Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle.

Genes and childhood leukemia.

Leukemia is a heterogeneous hematologic malignancy originating from a multipotent hematopoietic stem cell. It ranks among the commonest cancers in childhood and is characterized by excessive proliferation and differentiation block. The process of leukemogenesis is governed by genetic changes at both the cytogenetic and molecular level. According to numerous analyses, a large spectrum of mutations and rearrangements underlying the disease affect essential cellular transduction pathways, genes ensuring proper course of hematopoiesis, oncogenes, tumor suppressors and apoptosis regulators. Common lesions include translocations to T cell receptor (TCR) loci in T-lineage acute lymphoblastic leukemia (T-ALL), mutations of transcription factors regulating B-lineage development and cell maturation in B-lineage acute lymphoblastic leukemia (B-ALL) (PAX5, TCF3, EBF1, etc.), aberrational disruption of genes coding for transcription factors and coactivators in acute myeloid leukemia (AML) (e.g. CBF) or BCR-ABL1 fusion and activation of multiple kinases in chronic myeloid leukemia (CML). These alterations severely impair cell function. Broadening knowledge of the genetic background gives an insight into the pathobiology of a disease and allows for a better understanding of it. An appropriate investigation of genomic events yields diagnostic, prognostic and therapeutic implications. Broadening knowledge of the pathogenesis of leukemia seems to be a promising contribution to precise stratification of patients, reducing the toxicity and adverse effects caused by medical intervention, treatment personalization and introduction of targeted therapy accessible to a wide range of patients.