The explosion of knowledge in the past decade on the role of small double stranded (ds)RNAs in regulating gene expression has revolutionized our understanding of the basic biologic processes, as well as opened new ways to treat several human diseases. The Nobel Prize awarded just 7 years after the discovery of RNA interference (RNAi) highlights the tremendous potential of this phenomenon. Small RNAs involved in RNAi come in different forms, for example micro RNA (miRNA), siRNA, shRNA and piRNA. miRNAs generated from endogenously encoded genes are conserved from plants to mammals and serve to critically regulate gene expression to control almost all cell-fate decisions. RNAi also plays an essential role as a natural antiviral mechanism in plants and worms. Here, siRNAs generated by processing of long dsRNA (e.g., generated during viral infection) act to silence viral replication. In plants and worms, the siRNAs generated can also be amplified by the host enzyme RNA-dependent RNA polymerase (RdRP) and are transported from cell to cell, in a process known as spreading, to protect uninfected cells. Recent studies suggest that RNAi spreading can also occur in flies (Drosophila), although they lack RdRP. In keeping with the evolutionary relationship between the host and parasite, the viruses in turn encode proteins that can suppress the RNA-silencing response at multiple levels.
Although it is thought that siRNAs are not generated in mammals in response to viral infection, presumably because the interferon system and adaptive immunity have taken over the host response, several mammalian viruses also encode RNA silencing suppressors (RSS). Recent discoveries have also made it clear that the RNAi pathway is indeed also intricately involved in viral infections of mammals. For example, several DNA viruses, such as members of the Herpesviridae family, encode viral miRNAs that use the cellular RNAi machinery to target viral or cellular mRNAs, and these viral miRNAs critically control lytic/latent infection status and can also influence virus-induced oncogenesis. Furthermore, certain cellular miRNAs can directly target the viral genome to inhibit or enhance viral replication, and some viral infections are associated with specific alterations in the cellular miRNA expression profile.
The existence of RNAi machinery also makes it possible for exotic designer siRNA or shRNA to be used for silencing virtually any gene of interest in a sequence-specific manner. Ever since externally introduced ds-siRNAs were shown to silence specific gene expression in mammalian cells without inducing the harmful interferon response, there has been tremendous interest in applying them as potential novel drugs for the treatment of a variety of diseases, such as cancer, neurodegenerative diseases and viral infections. Several clinical studies are already evaluating siRNAs for human therapy. In proof-of-principal studies, several in mice or primate models, many viral infections have been effectively suppressed using siRNA. While synthetic siRNAs appear to be well suited for acute infections, endogenous generation of siRNAs via shRNA-expressing plasmid or viral vectors appears to be ideal for the long-term expression required for inhibiting chronic viral infections. With the advance in knowledge of cellular miRNA targets, miRNA mimics and antagomirs are also being added to the list of potential therapeutic tools and, in fact, anti-miR-122 is being evaluated for anti-hepatitis C virus (HCV) therapy. The main breakthrough that would enable siRNA therapy is the development of specific delivery methods. Rapid advances are being made in this field as well, with the development of targeted delivery approaches and improved nanoparticle formulations. Viruses, especially RNA viruses such as HIV-1, can rapidly generate escape mutants that can render a given siRNA ineffective but this can be overcome by using multiplexed siRNAs targeting several conserved regions in the viral genome, as well as combining with cellular targets essential for viral entry or replication. Multiplexed shRNA expression in a miRNA backbone in a viral vector is an example of the latest advancement in the field. With the current pace of discovery, it is only to be expected that siRNA/miRNA therapeutics are likely to become the routine treatment strategy in the next 5–10 years.
The book ‘RNA Interference and Viruses: Current Innovations and Future Trends’, edited by Miguel Angel Martinez, is a timely and well-compiled book, authored by several distinguished scientists who have made significant contributions to this important area of emerging research. The book consists of 11 chapters dealing with various aspects of the relevance of RNAi to viral infections in plant, insect and mammalian cells.
Chapter 1 focuses on RNAi as an antiviral mechanism in insects. Besides a background on RNAi, this chapter describes in detail the importance of RNAi as an essential natural mechanism to curtail viral infection in Drosophila and other insect species, as well as describing how viruses have evolved mechanisms to suppress the RNAi response to propagate themselves. The authors also discuss how even in the absence of RdRP in Drosophila (RdRP is thought to be essential for systemic spreading in plants and worms), RNAi spreads during viral infection to protect neighboring uninfected cells by endocytosis of viral dsRNA.
Chapter 2 describes how plant viruses suppress RNAi. RNA silencing is a natural antiviral defense mechanism in plants and to escape from this RNA silencing-based defense, plant viruses have evolved several strategies to suppress RNAi. These RNA-silencing suppressors act at various stages, starting from the generation of siRNAs from long viral dsRNA to blocking dicer or argonaute protein activity to sequestration of si/miRNAs. The molecular details of suppressor activities are elegantly described.
Chapter 3 deals with miRNAs encoded by mammalian viruses. Several DNA viruses, notably members of the Herpesviridae family, encode viral miRNAs during specific stages of the viral life cycle. The viral miRNAs can target viral genes to regulate latency/lytic infection, reduce viral antigen loads to suppress host immunity and/or contribute to oncogenicity by targeting cellular genes like transcription factors. The nature and molecular details on the role of these miRNAs are described in depth.
Chapter 4 describes how cellular miRNAs in mammalian cells may target viral genes to modulate viral replication as well as describing the RSS activity of certain mammalian virus-encoded proteins. The authors also discuss the multifunctionality of RSS proteins and the intricate relationship between the mammalian interferon pathway and RSS activity.
Chapter 5 discusses the unusual role of the liver-specific miR-122 in enhancing HCV replication. The mechanisms of interaction of miR-122 with the target sequences in the HCV untranslated region and its effect on HCV RNA replication and translation are discussed with great insight. The authors also discuss the identified natural cellular targets of miR-122, their role in cholesterol biosynthesis and hepatocellular carcinogenesis. In addition, recent efforts to neutralize this miRNA to modulate cholesterol levels and HCV replication in animal models are described in detail.
Chapter 6 describes how mammalian viruses can resist RNAi. Mutational escape from artificially induced RNAi, modulation of natural RNAi (miRNA pathway) via virally encoded RSS proteins and virus-induced alterations in cellular miRNA profile are discussed.
Chapter 7 discusses the efforts made so far in using RNAi to suppress viral infections with special reference to HIV-1. Problems involved in using RNAi therapeutics in chronic infections such as the generation of escape mutants and efforts to overcome this by using highly conserved target regions and multiplexing, along with targeting cellular factors involved in the viral life cycle, are all discussed in depth. Also discussed are the merits of externally applied siRNA versus endogenously generated shRNA as antiviral agents and efforts made to deliver these reagents to HIV‑susceptible cell types.
Chapter 8 looks into the use of siRNA and shRNA vectors to suppress hepatitis B virus infections. Measures to overcome the lack of a suitable small-animal model to test RNAi effectiveness in vivo and advances in delivering siRNA via nonviral vectors as well as the use of adeno and adeno-associated vectors to deliver shRNA to the liver to suppress hepatitis B virus are detailed.
Chapter 9 discusses the use of RNAi in the research and therapy of HCV infection. The authors describe how the use of RNAi to investigate the viral and host factors involved in HCV infection has led to the identification of several potential new drug targets. The development of RNAi-based therapeutics to suppress HCV infection is also described.
Chapter 10 describes the application of RNAi to inhibit respiratory viruses. Special emphasis is paid to detailing efforts to use siRNAs in the treatment of respiratory syncytial virus and parainfluenza virus where intranasal administration of naked siRNAs appears to effectively reduce virus replication. Some of these siRNAs are in an advanced clinical trial.
Chapter 11, the final chapter, details the use of viral vectors to induce RNAi. The different viral vectors used to inhibit different diseases such as cancer, neurological disorders and viral infection in animal models are described. In addition, the limitations of RNAi, such as toxicities induced by activation of the interferon pathway, off-target gene silencing and saturation of miRNA machinery are discussed.
Although some sections appear to be repetitive (probably unavoidable in a large compilation from authors around the world), the book addresses a range of important fundamental issues that may impact on the development of RNAi-based therapies against several human diseases. It provides a solid introduction to the general concepts in the field of RNAi, how viruses modulate RNAi responses, as well as issues involved in using RNAi as antiviral therapy. Thus, this book will be useful to a wide range of readers – from basic science students, to RNAi researchers, to virologists, to investors in drug-development companies. Considering the extraordinarily rapid progress being made in the field (several newer delivery methods that can reduce the siRNA requirement by one-to-two orders of magnitude have been described since this book was written), the authors will probably feel compelled to update this book on a regular basis.
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.