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.

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.

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.

Defective interfering particles (DIPs) represent promising antivirals against influenza A virus (IAV), yet strategies to further enhance their antiviral activity remain largely unexplored. Here, we engineered a next-generation “OP7” DIP by truncating the viral interferon (IFN) antagonist non-structural protein 1 (NS1) to enhance IFN-mediated antiviral activity. A previously validated mathematical multiscale model predicted that loss of NS1 function would enhance antiviral activity, providing a mechanistic rationale for this design. We engineered “OP7-trNS1” DIP, established a cell culture-based production process yielding infectious virus-free material, and evaluated its antiviral activity in human lung epithelial cells in vitro . OP7-trNS1 inhibited IAV replication by approx. two orders of magnitude more effectively than the parental OP7 DIP while inducing a stronger IFN response, consistent with the model predictions. Together, these findings demonstrate that reducing viral IFN antagonism is a viable strategy for rationally enhancing DIP antiviral activity and establish OP7-trNS1 as a promising candidate for further preclinical development.