Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems

RNA silencing is a host defense mechanism that limits the accumulation and spread of viruses in infected plants. Correspondingly, plant viruses encode suppressors of silencing. In the positive-strand RNA virus Tobacco rattle virus (TRV), the suppressor of silencing is a 16-kDa (16K) protein encoded by RNA1. The suppressor action of the 16K protein is transient and weaker than that of the P19 suppressor, encoded by tomato bushy stunt virus. Mutant TRV that does not produce its suppressor, unlike other suppressor-defective viruses, is competent to accumulate and spread systemically in the infected plant. However, this mutant virus does not exhibit the transient invasion of the meristem that is characteristic of the wild-type virus. Based on this analysis, we propose that the 16K suppressor of silencing allows TRV to transiently invade the meristem. Our data are consistent with a mechanism of long-term meristem virus exclusion that is dependent on a transient invasion of the meristem early in the infection cycle. This novel mechanism of meristem exclusion may be associated with the phenomenon of recovery in virus-infected plants in which upper leaves have little or no virus and are immune to secondary infection by the same virus.     

重组马铃薯X病毒载体介导的烟草γ-微管蛋白基因沉默

用重组马铃薯X病毒(potato virus X,PVX)载体将γ-微管蛋白反义基因导入烟草(Nicotiana tabacum var.Samsun NN),得到了γ-微管蛋白基因沉默的烟草植株。它与侵染PVX空载体的正对照相比有明显的差异:不同形态的叶片分层交替生长;到生殖期所有的花苞都提前脱落;小孢子不能发育到四分体阶段。沉默的形态反应主要起始于顶端幼嫩组织。在沉默过程中除存在基因沉默及恢复现象外还出现靶基因水平的陡然升高,甚至有时会明显反超过正常对照水平。     

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference

RNA interference (RNAi) might be an efficient antiviral therapy for some obstinate illness. Herein, a retrovirus-based RNAi system was developed to drive expression and delivery of Hepatitis B virus (HBV)-specific short hairpin RNA (shRNA) in HepG2 cells. The levels of HBsAg and HBeAg and that of HBV mRNA were dramatically decreased by this RNAi system in HepG2 cells transfected with Topo-HBV plasmid. Retrovirus-based RNAi thus may be useful for therapy in HBV and other viral infections and provide new clues for prophylactic vaccine development.     

RNA silencing pathways of plants: silencing and its suppression by plant DNA viruses

RNA silencing refers to processes that depend on small (s)RNAs to regulate the expression of eukaryotic genomes. In plants, these processes play critical roles in development, in responses to a wide array of stresses, in maintaining genome integrity and in defense against viral and bacterial pathogens. We provide here an updated view on the array of endogenous sRNA pathways, including microRNAs (miRNAs), discovered in the model plant Arabidopsis, which are also the basis for antiviral silencing. We emphasize the current knowledge as well as the recent advances made on understanding the defense and counter-defense strategies evolved in the arms race between plants and DNA viruses on both the nuclear and the cytoplasmic front. This article is part of a Special Issue entitled: MicroRNA's in viral gene regulation.     

Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference

To overcome the low efficiency of agronomic protection from maize dwarf mosaic disease, susceptible maize inbred line was transformed by Agrobacterium tumefaciens harboring hpRNA expression vectors containing inverted-repeat sequences of different lengths targeting coat protein gene (CP) of maize dwarf mosaic virus (MDMV). After PCR screening and Southern blotting, the flanking sequences of the integration sites were amplified by thermal asymmetric interlaced PCR (TAIL-PCR) and used for analysis of T-DNA integration patterns. The T? plant lines were evaluated for their MDMV resistance in field inoculation trials under two environments. Of the nineteen T? plant lines positive in Southern blotting, six were evaluated as resistant to MDMV, and four of them had resistance non-significantly different from the highly resistant control "H9-21", while the resistance of the other eleven was proved to be significantly improved when compared to their non-transformed parent line. These improvements in MDMV resistance were verified by the relative amount of virus CP gene expression measured by quantitative real time PCR. Comparing the results of Southern blotting and TAIL-PCR analysis, different integration patterns of one or two copies of the inverted-repeat sequences were identified from non-repetitive and repetitive sequences of the maize genome. The MDMV resistance mediated by RNA interference is relative to the length of the inverted-repeat sequence, the copy number of T-DNA integration and the repeatability of integration sites. A longer hpRNA expression construct shows more efficiency than a shorter one.     

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.     

A plant virus vector for systemic expression of foreign genes in cereals

Inserts bearing the coding sequences of NPT II and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18-30 days post-inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT-PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS-NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.     

病毒siRNA介导的RNA干扰途径与机制

RNA干扰(RNAi)是真核生物体内重要的基因表达调控方式之一.RNAi的一种原始的作用是帮助生物体抵抗病毒,早期的研究表明无脊椎动物可以利用RNAi抵抗病毒,但是哺乳动物是否存在这一机制一直存在争议.最新的研究发现了哺乳动物RNAi抗病毒的强有力的证据,并且研究人员认为,这是一种之前被忽视的、全新的免疫途径.值得注意...     

A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference

Allele-specific silencing by RNA interference (ASP-RNAi) is an atypical RNAi that is capable of discriminating target alleles from non-target alleles, and may be therapeutically useful for specific inhibition of disease-causing alleles without affecting their corresponding normal alleles. However, it is difficult to design and select small interfering RNA (siRNAs) that confer ASP-RNAi. A major problem is that there are few appropriate measures in determining optimal allele-specific siRNAs. Here we show two novel formulas for calculating a new measure of allele-discrimination, named “ASP-score”. The formulas and ASP-score allow for an unbiased determination of optimal siRNAs, and may contribute to characterizing such allele-specific siRNAs.     

Autocidal control of ticks by silencing of a single gene by RNA interference

Ticks impact human and animal health worldwide and new control methods are needed to circumvent drawbacks of tick control by acaricide application including selection of drug resistant ticks and environmental pollution. Using RNA interference we silenced the expression of a single gene, subolesin, and produced ticks with diminished reproductive performance and prevented successful mating and production of viable offspring. We propose a sterile acarine technique (SAT) for reduction of tick populations by release of subolesin-silenced ticks. Conservation of subolesin among tick species suggests that SAT may be useful for control of many medically and economically important tick species.     

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells

Background

Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells.

Methodology/Principal Findings

HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells.

Conclusions/Significance

Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.     

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80-90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.     

RUSH and CRUSH: A rapid and conditional RNA interference method in mice

RNA interference (RNAi) is a powerful approach to phenocopy mutations in many organisms. Gold standard conventional knock‐out mouse technology is labor‐ and time‐intensive; however, off‐target effects may confound transgenic RNAi approaches. Here, we describe a rapid method for conditional and reversible gene silencing in RNAi transgenic mouse models and embryonic stem (ES) cells. RUSH and CRUSH RNAi vectors were designed for reversible or conditional knockdown, respectively, demonstrated using targeted replacement in an engineered ROSA26^lacZ ES cell line and wildtype V6.5 ES cells. RUSH was validated by reversible knockdown of Dnmt1 in vitro. Conditional mouse model production using CRUSH was expedited by deriving ES cell lines from Cre transgenic mouse strains (nestin, cTnnT, and Isl1) and generating all‐ES G₀ transgenic founders by tetraploid complementation. A control CRUSH^GFP RNAi mouse strain showed quantitative knockdown of GFP fluorescence as observed in compound CRUSH^GFP, Ds‐Red Cre‐reporter transgenic mice, and confirmed by Western blotting. The capability to turn RUSH and CRUSH alleles off or on using Cre recombinase enables this method to rapidly address questions of tissue‐specificity and cell autonomy of gene function in development. genesis 52:39–48, 2014. © 2013 Wiley Periodicals, Inc.