Mathematical model calibrated to in vitro data predicts mechanisms of antiviral action of the influenza defective interfering particle "OP7".

Defective interfering particles (DIPs) are regarded as potent broad-spectrum antivirals. We developed a mathematical model that describes intracellular co-infection dynamics of influenza standard virus (STV) and "OP7", a new type of influenza DIP discovered recently. Based on experimental data from in vitro studies to calibrate the model and confirm its predictions, we deduce OP7's mechanisms of interference, which were yet unknown. Simulations suggest that the "superpromoter" on OP7 genomic viral RNA enhances its replication and results in a depletion of viral proteins. This reduces STV genomic RNA replication, which appears to constitute an antiviral effect. Further, a defective viral protein (M1-OP7) likely causes the deficiency of OP7's replication. It appears unable to bind to genomic viral RNAs to facilitate their nuclear export, a critical step in the viral life cycle. An improved understanding of OP7's antiviral mechanism is crucial toward application in humans as a prospective antiviral treatment strategy.

Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs.

The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2'-deoxy-2'-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%-88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6-1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.

Universal and strain specific structure features of segment 8 genomic RNA of influenza A virus-application of 4-thiouridine photocrosslinking.

RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.

Conserved Structural Motifs of Two Distant IAV Subtypes in Genomic Segment 5 RNA.

The functionality of RNA is fully dependent on its structure. For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging. Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important. Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology. However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation. In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis. Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses. A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed. These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.

Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle-Structure, Interactions, and Implications for the Emergence of New Strains.

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA-RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

RNA Secondary Structure Motifs of the Influenza A Virus as Targets for siRNA-Mediated RNA Interference.

The influenza A virus is a human pathogen that poses a serious public health threat due to rapid antigen changes and emergence of new, highly pathogenic strains with the potential to become easily transmitted in the human population. The viral genome is encoded by eight RNA segments, and all stages of the replication cycle are dependent on RNA. In this study, we designed small interfering RNA (siRNA) targeting influenza segment 5 nucleoprotein (NP) mRNA structural motifs that encode important functions. The new criterion for choosing the siRNA target was the prediction of accessible regions based on the secondary structure of segment 5 (+)RNA. This design led to siRNAs that significantly inhibit influenza virus type A replication in Madin-Darby canine kidney (MDCK) cells. Additionally, chemical modifications with the potential to improve siRNA properties were introduced and systematically validated in MDCK cells against the virus. A substantial and maximum inhibitory effect was achieved at concentrations as low as 8 nM. The inhibition of viral replication reached approximately 90% for the best siRNA variants. Additionally, selected siRNAs were compared with antisense oligonucleotides targeting the same regions; this revealed that effectiveness depends on both the target accessibility and oligonucleotide antiviral strategy. Our new approach of target-site preselection based on segment 5 (+)RNA secondary structure led to effective viral inhibition and a better understanding of the impact of RNA structural motifs on the influenza replication cycle.