Poliovirus Escape from RNA Interference: Short Interfering RNA-Target Recognition and Implications for Therapeutic Approaches

ABSTRACT Short interfering RNAs (siRNAs) directed against poliovirus and other viruses effectively inhibit viral replication. Although RNA interference (RNAi) may provide the basis for specific antiviral therapies, the limitations of RNAi antiviral strategies are ill defined. Here, we show that poliovirus readily escapes highly effective siRNAs through unique point mutations within the targeted regions. Competitive analysis of the escape mutants provides insights into the basis of siRNA recognition. The RNAi machinery can tolerate mismatches but is exquisitely sensitive to mutations within the central region and the 3′ end of the target sequence. Indeed, specific mutations in the target sequence resulting in G:U mismatches are sufficient for the virus to escape siRNA inhibition. However, using a pool of siRNAs to simultaneously target multiple sites in the viral genome prevents the emergence of resistant viruses. Our study uncovers the elegant precision of target recognition by the RNAi machinery and provides the basis for the development of effective RNAi-based therapies that prevent viral escape.

Download Full-text

Hepatitis C Virus Replicons Escape RNA Interference Induced by a Short Interfering RNA Directed against the NS5b Coding Region

Journal of Virology ◽

10.1128/jvi.79.11.7050-7058.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 7050-7058 ◽

Cited By ~ 138

Author(s):

Joyce A. Wilson ◽

Christopher D. Richardson

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Interference ◽

Point Mutations ◽

Subsequent Treatment ◽

Escape Mutant ◽

Target Sequence ◽

Short Interfering Rna ◽

Coding Region ◽

Interfering Rna

ABSTRACT RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.

Download Full-text

Human Immunodeficiency Virus Type 1 Escape Is Restricted When Conserved Genome Sequences Are Targeted by RNA Interference

Journal of Virology ◽

10.1128/jvi.02035-07 ◽

2007 ◽

Vol 82 (6) ◽

pp. 2895-2903 ◽

Cited By ~ 94

Author(s):

Karin Jasmijn von Eije ◽

Olivier ter Brake ◽

Ben Berkhout

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Interference ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Viral Escape ◽

Specific Gene ◽

Target Sequence ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but—in contrast to what is observed with nef—no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.

Download Full-text

Faculty Opinions recommendation of Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026122.322997 ◽

2005 ◽

Author(s):

Mark Westhusin

Keyword(s):

Rna Interference ◽

Target Recognition ◽

Short Interfering Rna ◽

Therapeutic Approaches ◽

Interfering Rna

Download Full-text

Faculty Opinions recommendation of DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029052.345437 ◽

2005 ◽

Author(s):

Andy Maule

Keyword(s):

Rna Interference ◽

Plant Cell ◽

Small Interfering Rna ◽

Interfering Rna

Download Full-text

Amphiphysin 1 Is Important for Actin Polymerization during Phagocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-04-0296 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4669-4680 ◽

Cited By ~ 30

Author(s):

Hiroshi Yamada ◽

Emiko Ohashi ◽

Tadashi Abe ◽

Norihiro Kusumi ◽

Shun-AI Li ◽

...

Keyword(s):

Rna Interference ◽

Sertoli Cells ◽

Small Interfering Rna ◽

Actin Polymerization ◽

Actin Dynamics ◽

Polymerization Process ◽

Amphiphysin 1 ◽

Phagocytic Cups ◽

Interfering Rna ◽

Constitutively Active

Amphiphysin 1 is involved in clathrin-mediated endocytosis. In this study, we demonstrate that amphiphysin 1 is essential for cellular phagocytosis and that it is critical for actin polymerization. Phagocytosis in Sertoli cells was induced by stimulating phosphatidylserine receptors. This stimulation led to the formation of actin-rich structures, including ruffles, phagocytic cups, and phagosomes, all of which showed an accumulation of amphiphysin 1. Knocking out amphiphysin 1 by RNA interference in the cells resulted in the reduction of ruffle formation, actin polymerization, and phagocytosis. Phagocytosis was also drastically decreased in amph 1 (−/−) Sertoli cells. In addition, phosphatidylinositol-4,5-bisphosphate–induced actin polymerization was decreased in the knockout testis cytosol. The addition of recombinant amphiphysin 1 to the cytosol restored the polymerization process. Ruffle formation in small interfering RNA-treated cells was recovered by the expression of constitutively active Rac1, suggesting that amphiphysin 1 functions upstream of the protein. These findings support that amphiphysin 1 is important in the regulation of actin dynamics and that it is required for phagocytosis.

Download Full-text

Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽

10.3390/cells10051187 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1187

Author(s):

Michael Wassenegger ◽

Athanasios Dalakouras

Keyword(s):

Dna Methylation ◽

Rna Interference ◽

Gene Silencing ◽

Plant Cells ◽

Transcriptional Gene Silencing ◽

Rnai Machinery ◽

Double Stranded Rna ◽

Post Transcriptional Gene Silencing ◽

Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

Download Full-text

Computational Design of Antiviral RNA Interference Strategies That Resist Human Immunodeficiency Virus Escape

Journal of Virology ◽

10.1128/jvi.79.3.1645-1654.2005 ◽

2005 ◽

Vol 79 (3) ◽

pp. 1645-1654 ◽

Cited By ~ 50

Author(s):

Joshua N. Leonard ◽

David V. Schaffer

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Interference ◽

Highly Active Antiretroviral Therapy ◽

Viral Evolution ◽

Regulation Of Gene Expression ◽

Computational Design ◽

Delivery Systems ◽

Target Sequence ◽

Therapeutic Success ◽

Immunodeficiency Virus

ABSTRACT Recently developed antiviral strategies based upon RNA interference (RNAi), which harnesses an innate cellular system for the targeted down-regulation of gene expression, appear highly promising and offer alternative approaches to conventional highly active antiretroviral therapy or efforts to develop an AIDS vaccine. However, RNAi is faced with several challenges that must be overcome to fully realize its promise. Specifically, it degrades target RNA in a highly sequence-specific manner and is thus susceptible to viral mutational escape, and there are also challenges in delivery systems to induce RNAi. To aid in the development of anti-human immunodeficiency virus (anti-HIV) RNAi therapies, we have developed a novel stochastic computational model that simulates in molecular-level detail the propagation of an HIV infection in cells expressing RNAi. The model provides quantitative predictions on how targeting multiple locations in the HIV genome, while keeping the overall RNAi strength constant, significantly improves efficacy. Furthermore, it demonstrates that delivery systems must be highly efficient to preclude leaving reservoirs of unprotected cells where the virus can propagate, mutate, and eventually overwhelm the entire system. It also predicts how therapeutic success depends upon a relationship between RNAi strength and delivery efficiency and uniformity. Finally, targeting an essential viral element, in this case the HIV TAR region, can be highly successful if the RNAi target sequence is correctly selected. In addition to providing specific predictions for how to optimize a clinical therapy, this system may also serve as a future tool for investigating more fundamental questions of viral evolution.

Download Full-text

Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

Journal of Experimental Medicine ◽

10.1084/jem.20110740 ◽

2011 ◽

Vol 208 (13) ◽

pp. 2599-2606 ◽

Cited By ~ 111

Author(s):

Whitney E. Purtha ◽

Thomas F. Tedder ◽

Syd Johnson ◽

Deepta Bhattacharya ◽

Michael S. Diamond

Keyword(s):

B Cells ◽

Plasma Cells ◽

Large Fraction ◽

West Nile Virus Infection ◽

Immune Serum ◽

Memory B Cells ◽

Viral Escape ◽

Variant Virus ◽

Escape Mutants

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) persist after clearance of infection, yet the specific and nonredundant role MBCs play in subsequent protection is unclear. After resolution of West Nile virus infection in mice, we demonstrate that LLPCs were specific for a single dominant neutralizing epitope, such that immune serum poorly inhibited a variant virus that encoded a mutation at this critical epitope. In contrast, a large fraction of MBC produced antibody that recognized both wild-type (WT) and mutant viral epitopes. Accordingly, antibody produced by the polyclonal pool of MBC neutralized WT and variant viruses equivalently. Remarkably, we also identified MBC clones that recognized the mutant epitope better than the WT protein, despite never having been exposed to the variant virus. The ability of MBCs to respond to variant viruses in vivo was confirmed by experiments in which MBCs were adoptively transferred or depleted before secondary challenge. Our data demonstrate that class-switched MBC can respond to variants of the original pathogen that escape neutralization of antibody produced by LLPC without a requirement for accumulating additional somatic mutations.

Download Full-text

Saccharomyces cerevisiae (Baker’s Yeast) as an Interfering RNA Expression and Delivery System

Current Drug Targets ◽

10.2174/1389450120666181126123538 ◽

2019 ◽

Vol 20 (9) ◽

pp. 942-952 ◽

Cited By ~ 9

Author(s):

Molly Duman-Scheel

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Interference ◽

Genetic Model ◽

Model Organism ◽

Rna Expression ◽

Commercial Development ◽

Rna Integrity ◽

Yeast Cultures ◽

Ideal System ◽

Interfering Rna

The broad application of RNA interference for disease prevention is dependent upon the production of dsRNA in an economically feasible, scalable, and sustainable fashion, as well as the identification of safe and effective methods for RNA delivery. Current research has sparked interest in the use of Saccharomyces cerevisiae for these applications. This review examines the potential for commercial development of yeast interfering RNA expression and delivery systems. S. cerevisiae is a genetic model organism that lacks a functional RNA interference system, which may make it an ideal system for expression and accumulation of high levels of recombinant interfering RNA. Moreover, recent studies in a variety of eukaryotic species suggest that this microbe may be an excellent and safe system for interfering RNA delivery. Key areas for further research and development include optimization of interfering RNA expression in S. cerevisiae, industrial-sized scaling of recombinant yeast cultures in which interfering RNA molecules are expressed, the development of methods for largescale drying of yeast that preserve interfering RNA integrity, and identification of encapsulating agents that promote yeast stability in various environmental conditions. The genetic tractability of S. cerevisiae and a long history of using this microbe in both the food and pharmaceutical industry will facilitate further development of this promising new technology, which has many potential applications of medical importance.

Download Full-text