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

Piasecka, Julita; Lenartowicz, Elzbieta; Soszynska-Jozwiak, Marta; Szutkowska, Barbara; Kierzek, Ryszard; Kierzek, Elżbieta

doi:10.1016/j.omtn.2019.12.018

Cited by 28 publications

(37 citation statements)

References 77 publications

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“…Modifications of siRNA may influence different stages of RNAinduced silencing complex (RISC) actions. 31,32 To examine whether the LNA-modification may influence the knockdown activity and to evaluate KK-46 transfection activity with siRNA, Vero E6 cells no significant off-target effects on the vRNA levels neither in cell lysates nor in supernatants. We found no significant difference in antiviral effects of unmodified siR-7 or LNA-modified siR-7-EM but complexes with siR-7-EM seemed to be more effective than siR-7 complexes which was also reflected by the fact that only the 21 µg dose of siR-7-EM gave significant results compared to siLuc, p < 0.05.…”

Section: Sir-7-em/peptide Dendrimer Kk-46 Complexes Exhibit Strong Anti-sars-cov-2 Activity In Vitromentioning

confidence: 99%

Silencing of SARS‐CoV‐2 with modified siRNA‐peptide dendrimer formulation

Khaitov

Nikonova

Shilovskiy

et al. 2021

Allergy

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Background: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo.Methods: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation -siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. Results:We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro.Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo.Conclusions: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients. | 2841KHAITOV eT Al.

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Section: Sir-7-em/peptide Dendrimer Kk-46 Complexes Exhibit Strong Anti-sars-cov-2 Activity In Vitromentioning

confidence: 99%

Silencing of SARS‐CoV‐2 with modified siRNA‐peptide dendrimer formulation

Khaitov

Nikonova

Shilovskiy

et al. 2021

Allergy

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“…Moreover, therapies depending on protein might quickly become useless due to the potential of RNA viruses to evade such drugs through evolved resistance [ 30 , 31 , 32 , 33 ]. Our previous studies concerning RNA structure contributed to the development of anti-influenza inhibitors directly targeting the RNA structural motifs that showed high antiviral potential when tested in cells [ 15 , 34 , 35 , 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

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

Michalak

Piasecka

Szutkowska

et al. 2021

Viruses

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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.

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“…Overall, we have demonstrated that NS mRNA secondary structures can affect viral replication in vivo. Previously, it was pointed out that regions of viral RNA featuring conserved secondary structures may be used as potential targets in new generations of antiviral therapy [12,39] or for the development of live vaccines [32]. However, considering the slight contribution of NS mRNA secondary structures to viral pathogenesis, these structures are unlikely to be significant pathogenicity factors, but are rather involved in the fine regulations of NS1 and NEP gene expression.…”

Section: Discussionmentioning

confidence: 99%

“…During the process of viral replication, positive-sense RNA is also synthesized, which is classified into a genomic complementary (+)RNA (cRNA) or messenger RNA (mRNA). Currently, the function of several secondary structures within promoter regions of vRNA and cRNA, and splice sites in the M and NS genes [12] are only partially understood.…”

Section: Introductionmentioning

confidence: 99%

Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

Baranovskaya

Sergeeva

Taraskin

et al. 2021

Microbiology Independent Research Journal (MIR Journal)

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The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

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

Cited by 28 publications

References 77 publications

Silencing of SARS‐CoV‐2 with modified siRNA‐peptide dendrimer formulation

Silencing of SARS‐CoV‐2 with modified siRNA‐peptide dendrimer formulation

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

Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

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