Suppression of HIV replication using RNA interference against HIV-1 integrase

RNA interference (RNAi) has become one of the most powerful and popular approach on gene silencing in clinical research study especially in virology due to the gene-specific suppression property of small interfering RNA (siRNA). In this report, we demonstrate that expression of vector-mediated small hairpin RNA (shRNA) against human immunodeficiency virus type 1 (HIV-1) integrase (IN), one of the three important enzymes in HIV infection by controlling the integration of viral RNA to host DNA, could suppress the protein synthesis of EGFP-tagged IN in HeLa cell model efficiently. Furthermore, we show that IN shRNA can successfully reduce the HIV particles production in 293T cells at the level similar to the positive control of HIV-1 tat shRNA. These results provide the therapeutic possibility of HIV replication using RNAi against HIV-1 integrase.     

Disturbance of the microRNA pathway by commonly used lentiviral shRNA libraries limits the application for screening host factors involved in hepatitis C virus infection

RNA interference (RNAi) is widely used as a screening tool for the identification of host genes involved in viral infection. Due to the limitation of raw small interfering RNA (siRNA), we tested two commonly used short hairpin RNA (shRNA) lentiviral libraries to identify host factors involved in hepatitis C virus (HCV) infection. It was found that these shRNA library vectors caused non-specific disturbance of HCV replication that was not due to toxicity or interferon response, but related to the high shRNA levels disturbing the endogenous microRNA biogenesis. The high shRNA levels achieved with these vectors reduced the levels of mature microRNAs, including miR-122 known to promote HCV replication. Our findings extend the caution of potential off-target effects of lentiviral shRNA libraries which appear unsuitable to screen microRNA regulated phenotypes, such as HCV replication.     

Human immunodeficiency virus type 1 escape from RNA interference

Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.     

Network based analysis of hepatitis C virus core and NS4B protein interactions

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. Here we attempt to further our understanding of the biological context of protein interactions in HCV pathogenesis, by investigating interactions between HCV proteins Core and NS4B and human host proteins. Using the yeast two-hybrid (Y2H) membrane protein system, eleven human host proteins interacting with Core and 45 interacting with NS4B were identified, most of which are novel. These interactions were used to infer overall protein interaction maps linking the viral proteins with components of the host cellular networks. Core and NS4B proteins contribute to highly compact interaction networks that may enable the virus to respond rapidly to host physiological responses to HCV infection. Analysis of the interaction networks highlighted enriched biological pathways likely influenced in HCV infection. Inspection of individual interactions offered further insights into the possible mechanisms that permit HCV to evade the host immune response and appropriate host metabolic machinery. Follow-up cellular assays with cell lines infected with HCV genotype 1b and 2a strains validated Core interacting proteins ENO1 and SLC25A5 and host protein PXN as novel regulators of HCV replication and viral production. ENO1 siRNA knockdown was found to inhibit HCV replication in both the HCV genotypes and viral RNA release in genotype 2a. PXN siRNA inhibition was observed to inhibit replication specifically in genotype 1b but not in genotype 2a, while SLC25A5 siRNA facilitated a minor increase in the viral RNA release in genotype 2a. Thus, our analysis can provide potential targets for more effective anti-HCV therapeutic intervention.     

Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.     

针对SARS冠状病毒重要蛋白的siRNA设计(英)

RNA干涉(RNA interference, RNAi)是一种特异性地导致转录后基因沉默的现象,在哺乳动物细胞中小分子干扰RNA双链体(small interfering RNA duplexes, siRNA duplexes)可以有效地诱导RNAi现象,为一些疾病的治疗开辟了新的途径.针对SARS冠状病毒(SARS coronavirus, SARS-CoV)中编码5个主要蛋白质的基因,用生物信息学的方法设计了348条候选siRNA靶标.在理论上,相应的siRNA双链体能特异地抑制SARS-CoV靶基因的表达,同时不会影响人体细胞基因的正常表达,这为进一步siRNA类药物的实验研究提供了理论基础.     

针对SARS冠状病毒重要蛋白的siRNA设计(英文)

NA干涉 (RNAinterference ,RNAi)是一种特异性地导致转录后基因沉默的现象 ,在哺乳动物细胞中小分子干扰RNA双链体 (smallinterferingRNAduplexes ,siRNAduplexes)可以有效地诱导RNAi现象 ,为一些疾病的治疗开辟了新的途径 .针对SARS冠状病毒 (SARScoronavirus ,SARS CoV)中编码 5个主要蛋白质的基因 ,用生物信息学的方法设计了3 48条候选siRNA靶标 .在理论上 ,相应的siRNA双链体能特异地抑制SARS CoV靶基因的表达 ,同时不会影响人体细胞基因的正常表达 ,这为进一步siRNA类药物的实验研究提供了理论基础     

Slipping through the door: HIV entry into the nucleus

HIV infection of non-dividing cellular targets like macrophages requires successful passage of the viral preintegration complex (PIC) across an intact nuclear envelope. Unique but redundant nuclear import signals reside within the HIV integrase, matrix, and Vpr proteins as well as the 'DNA flap'; these signals appear to facilitate PIC transport through the limiting nuclear pores. We discuss recent studies that have advanced our understanding of this key step in the HIV life cycle.     

小干扰RNA的研究进展

RNA干扰(RNA interference,RNAi)是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mRNA产生特异性降解,进而引起不同水平的基因沉默,其效应分子主要是小干扰RNA(siRNA)。siRNA是生物界普遍存在的一种抵御外来基因和病毒感染的基因调控方式,也是一种重要的研究工具。大量的研究工作致力于设计合理的siRNA片段用于基因功能研究,并将其作为一种治疗方法用于肿瘤、病毒性疾病等基因治疗以及药物靶向研究。因此本文对siRNA的作用机制、设计原则及其在临床应用中的缺点和解决方法进行综述。     

Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro

Two approaches have been developed to construct plasmids that mediate RNA interference to inhibit the replication and expression of HBV in 2.2.15 cell. The overlapping PCR extension and restriction enzyme-digestion were used to generate DNA fragments encoding designed shRNA based on sequences of ORF C of HBV genome. The pU6 derived vectors were constructed to develop plasmid based shRNA delivery systems termed pU6/HBVi. There were significant reductions in the expression of HBsAg and HBeAg between cells transfected with pU6/HBVi and control groups (as to HBsAg: P < 0. 01; and HBeAg: P < 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 x 10(7) to <5 x 10(2) copies with or without pU6/HBVi. These results suggested that shRNA delivery by recombinant plasmids harboring shRNA encoding DNA fragment of interest generated either by overlapping PCR extension or restriction enzyme-digestion, could inhibit expressions of viral proteins and reduce viral replications. The pU6 derived plasmids might be a useful shRNA delivery system in mammalian cells. In addition, we found siRNA based on stealth 2311 was a potent RNAi target of HBV genome.     

小干扰RNA的研究进展

RNA干扰（RNA interference,RNAi）是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mR_NA产生特异性降解,进而引起不同水平的基因沉默。其效应分子主要是小干扰RNA（siRNA）。siRNA是生物界普遍存在的一种抵御外来基因和病毒感染的基因调控方式,也是一种重要的研究工具。大量的研究工作致力于设计合理的siRNA片段用于基因功能研究,并将其作为一种治疗方法用于肿瘤、病毒性疾病等基因治疗以及药物靶向研究。因此本文对siRNA的作用机制、设计原则及其在临床应用中的缺点和解决方法进行综述。     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

Photosensitizing carrier proteins for photoinducible RNA interference

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.