RNA Viruses and RNAi: Quasispecies Implications for Viral Escape

Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi) acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens.     

针对HBV—P基因的RNA干扰抑制乙型肝炎病毒的研究

目的构建针对乙型肝炎病毒P基因编码区、能在体内转录产生发夹状小干扰RNA（siRNA）的表达载体psiHBV／p,观察RNA干扰对HBsAg、HBeAg及乙型肝炎病毒DNA复制的抑制作用。方法针对HBV—P基因区特异序列,构建siRNA的表达载体psiHBV／p。采用脂质体介导方法将其与1．3倍HBV真核表达质粒pHBV1．3共转染HepG2细胞。分别于转染后24小时、48小时、72小时用ELISA法对HepG2细胞培养上清液进行HBsAg、HBeAg的检测;于转染后72小时通过FQ-PCR法分析RNA干扰作用对HBVDNA的抑制效果。结果成功构建了针对HBV—P基因区的siRNA的真核表达重组体psiHBV／p,并发现它能明显抑制HBsAg及HBeAg的分泌,转染后第二天抑制率达高峰,分别为84％、65％。FQ—PCR结果也证实了转染72小时后,随psiHBV／p比例的升高,其对HBV DNA的抑制作用也随之增加。结论成功构建的psiHBV／p,它能在体内持续转录产生针对P基因转录体的发夹状siRNA;在细胞水平上,体内转录产生的、针对乙型肝炎病毒P基因区特异序列的siRNA对共转染的重组载体pHBV1．3有显著和特异的抑制作用。     

Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response

BACKGROUND & AIMS: Persistent infection with hepatitis C virus (HCV) leads to chronic hepatitis and hepatocellular carcinoma (HCC). RNA interference (RNAi) may act as a host antiviral response against viral RNA. METHODS: The effects of RNAi on both the replicative intermediates and the internal ribosome entry site (IRES) of HCV were studied by using HCV-related short interfering RNA (siRNA) detection assay. The mechanism that permits HCV to escape RNAi was studied by using RNAi assay materials. RESULTS: These studies demonstrate that the Dicer, an RNase enzyme that generates short siRNA, can target and digest both the IRES and the replicative intermediate of HCV into siRNA of approximately 22 nucleotides. Further studies also show that Dicer can inhibit the replication of the HCV subgenomic replicon. However, the HCV core protein inhibits this RNAi and rescues the replication of the HCV subgenomic replicon through a direct interaction with Dicer. CONCLUSIONS: RNAi is a limiting factor for HCV infection, and the core protein suppresses the RNA silencing-based antiviral response. This ability of the core protein to counteract the host defense may lead to a persistent viral infection and may contribute to the pathogenesis of HCV.     

针对HBV X基因的siRNA抑制HepG2细胞HBV的复制和表达

目的构建能转录产生针对乙型肝炎病毒（HBV）X基因转录体的小干扰RNA（siRNA）转录载体pSIH—BV／X,研究其在体外对HBV复制和抗原表达的抑制作用。方法将构建成功的pSIHBV／X与HBV1．3倍体真核表达质粒pHBV1．3共转染HepG2细胞，转染后24、48、72h检测上清液中HBsAg、HBeAg的变化，并检测72h时HBV DNA的变化。结果成功构建了针对HBVX基因转录体的siRNA表达载体pSIHBV／X，并发现它能抑制HBsAg、HBeAg的分泌，抑制高峰在72h．抑制率分别是64％和61％，而无关对照序列的siRNA无此作用。荧光定量PCR证实HBV DNA的复制亦受到抑制，抑制率31％。结论针对HBVX基因转录体的siRNA在体外具有抑制HBV复制和抗原表达的作用。     

短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

目的研究载体表达的短发夹状双链RNA（shRNA）对丙型肝炎病毒（HCV）IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体（pIRES—GFP）和虫荧光索酶表达载体（p5′ UTR—Luc），以及针对HCV IRES的shRNA表达载体（pshRNA-HCV）。共转染HepG2细胞，于转染后24、48、72h观察绿色荧光的强弱，用Western blot检测绿色荧光蛋白的表达，半定量逆转录聚合酶链反应法检测GFP的mRNA水平。双荧光索酶系统检测虫荧光索酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组，GFP蛋白表达量及虫荧光素酶活性降低60％～70％，半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平，该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。     

Possibilities for RNA interference in developing hepatitis C virus therapeutics

The discovery and characterization of the RNA interference (RNAi) pathway has been one of the most important scientific developments of the last 12 years. RNAi is a cellular pathway wherein small RNAs control the expression of genes by either degrading homologous RNAs or preventing the translation of RNAs with partial homology. It has impacted basic biology on two major fronts. The first is the discovery of microRNAs (miRNAs), which regulate almost every cellular process and are required for some viral infections, including hepatitis C virus (HCV). The second front is the use of small interfering RNAs (siRNAs) as the first robust tool for mammalian cellular genetics. This has led to the identification of hundreds of cellular genes that are important for HCV infection. There is now a major push to adapt RNAi technology to the clinic. In this review, we explore the impact of RNAi in understanding HCV biology, the progress in design of RNAi-based therapeutics for HCV, and remaining obstacles.     

Host RNA circles and the origin of hepatitis delta virus

Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases.In some cases,such circles actually bind host microRNAs.Such depletion of available microRNAs appears to have biological roles;for instance,in homeostasis and disease.These findings regarding host RNA circles support a speculative reappraisal of the origin and mode of replication of hepatitis delta virus,hepatitis delta virus(HDV),an agent with a small circular RNA genome;specifically,it is proposed that in hepatocytes infected with hepatitis B virus(HBV),some viral RNA species are processed to circular forms,which by a series of chance events lead to an RNA that can be both replicated by host enzymes and assembled,using HBV envelope proteins,to form particles some of which are infectious.Such a model also may provide some new insights into the potential pathogenic potential of HDV infections.In return,new insights into HDV might provide information leading to a better understanding of the roles of the host RNA circles.     

A randomized,double-blind,placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively (P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients (P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.     

siRNA for Influenza Therapy

Influenza virus is one of the most prevalent and ancient infections in humans. About a fifth of world's population is infected by influenza virus annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised. In the US alone, influenza outbreaks lead to roughly 30,000 deaths each year. Current vaccines and anti-influenza drugs are of limited use due to high mutation rate of the virus and side effects. In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen. Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication. The siRNAs outperform traditional small molecule antivirals in a number of areas, such as ease of design, modest cost, and fast turnaround. Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism, which is reviewed here.     

DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.     

Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma

Primary liver cancer is the fifth most common malignancy in the world and is a leading cause of cancer-related mortality. Available treatment for hepatocellular carcinoma （HCC）, the commonest primary liver cancer, is rarely curative and there is a need to develop therapy that is more effective. Specific and powerful gene silencing that can be achieved by activating RNA interference （RNAi） has generated enthusiasm for exploiting this pathway for HCC therapy. Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus （HBV） and hepatitis C virus （HCV）. Proof of principle studies have demonstrated promising results, and an early clinical trial assessing RNAi-based HBV therapy is currently in progress. Although the data augur well, there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized. Particularly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.     

Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs’ effects on their host.     

Opportunities for treating chronic hepatitis B and C virus infection using RNA interference

Activating the RNA interference (RNAi) pathway to achieve silencing of specific genes is one of the most exciting new developments of molecular biology. A particularly interesting use of this technology is inhibition of defined viral gene expression. In this review, we discuss the potential application of RNAi to treatment of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Globally, these hepatotropic viruses are the most important causes of cirrhosis and liver cancer. Available treatments have their limitations, which makes development of novel effective RNAi-based therapies for HBV and HCV especially significant. Several investigations carried out in vitro and in vivo are summarized, which demonstrate proof of principle that HBV and HCV can be inhibited by RNAi activators. Challenges facing further development of this technology to a stage of clinical application are discussed.     

RNA干扰在动物体内抗乙型肝炎病毒的效果

目的以乙型肝炎病毒(HBV)C区为靶位,动物实验体内观察RNA干扰抗HBV的效果。方法以流体动力学法建立HBV感染的动物模型,将pcDNA 3．1-HBV和体外细胞实验证明有效的小干扰 RNA(siRNA)尾静脉共注射BALB／c小鼠;用时间分辨免疫荧光分析法检测小鼠血清中乙型肝炎表面抗原(HBsAg)水平,用荧光定量聚合酶链反应法检测血清HBV DNA水平,用逆转录聚合酶链反应法检测 HBV C-mRNA,用免疫组织化学法检测肝组织HBsAg和乙型肝炎核心抗原。结果在小鼠体内,siRNA 能有效抑制HBV的复制和表达,干扰效果至少持续3 d。结论靶向HBV C区的siRNA在动物体内能有效抗HBV。     

Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses

Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.     

Sustained delivery of siRNAs targeting viral infection by cell-degradable multilayered polyelectrolyte films

Gene silencing by RNA interference (RNAi) has been shown to represent a recently discovered approach for the treatment of human diseases, including viral infection. A major limitation for the success of therapeutic strategies based on RNAi has been the delivery and shortlasting action of synthetic RNA. Multilayered polyelectrolyte films (MPFs), consisting of alternate layer-by-layer deposition of polycations and polyanions, have been shown to represent an original approach for the efficient delivery of DNA and proteins to target cells. Using hepatitis C virus infection (HCV) as a model, we demonstrate that siRNAs targeting the viral genome are efficiently delivered by MPFs. This delivery method resulted in a marked, dose-dependent, specific, and sustained inhibition of HCV replication and infection in hepatocyte-derived cells. Comparative analysis demonstrated that delivery of siRNAs by MPFs was more sustained and durable than siRNA delivery by standard methods, including electroporation or liposomes. The antiviral effect of siRNA-MPFs was reversed by a hyaluronidase inhibitor, suggesting that active degradation of MPFs by cellular enzymes is required for siRNA delivery. In conclusion, our results demonstrate that cell-degradable MPFs represent an efficient and simple approach for sustained siRNA delivery targeting viral infection. Moreover, this MPF-based delivery system may represent a promising previously undescribed perspective for the use of RNAi as a therapeutic strategy for human diseases.     

Long‐term RNA interference and its application to hepatitis B virus

RNA interference (RNAi) is an ancient defensive mechanism in eukaryotes to control gene expressing and defend their genomes from foreign invaders. It refers to the phenomenon that double‐stranded RNA results in the sequence‐specific silencing of target gene expression. Although it was documented in a relatively short time ago, intensive research has facilitated making its mechanism clear. Researchers have found that it was a powerful tool for analyzing the functions of genes and treating tumors, infectious diseases and genetic abnormalities that are associated with a dominant gene defect. However, delivery in vivo, low blood stability and poor intracellular uptake present significant challenges for the development of RNAi reagents in clinical use. Thus, long‐term inducible RNAi was designed. There are hundreds of millions of hepatitis B virus (HBV) carriers in the world at present, a portion of whom will lose their lives after several years due to chronic complications such as cirrhosis, hepatocellular carcinomas or both. Although a preventive vaccine is now available, the present therapeutic options for chronically infected patients are limited and of low efficiency. Admittedly, to date most RNAi experiments have been done in vitro, but it is hoped that they may be developed into a therapeutic strategy for HBV in the near future. In this article the principles and construction of long‐term RNA are discussed. Its therapeutic potentiality and attention to the potential hazards will also outlined. We conclude that this ancient defensive mechanism can be recruited as a powerful weapon in the fight against HBV.