Properties of Antigenomic HDV Ribozyme Consisting of Two RNA Chains

B:LS ribozyme, a trans-variant of naturally occurring HDV ribozyme, has been constructed. The ribozyme consists of a substrate-containing LS chain and a catalytic B chain and differs from previously constructed trans-ribozymes in the length and nucleotide sequence of its oligonucleotide chains (33 and 34 bp, respectively). The chains readily associate with each other at room temperature, at which the LS cleavage reaction is negligible, which makes it possible to investigate association of the intact chains. At the same time, the self-cleavage rate constant for the trans-ribozyme B:LS at 50°C is close to those for the previously studied permuted cis-ribozymes, especially the LSB variant. In addition, the dependence of trans-ribozyme on reaction conditions (Mg²⁺ concentration, pH, and temperature) resembled that of cis-ribozyme. Similar to other trans-ribozymes, B:LS ribozyme demonstrated the ability for multiple turnover of the B strand with an excess of the substrate LS chain. The kinetic model of the self-cleavage reaction for B:LS is presented at http://www.cardio.ru/labgen/RZ_r.html. Taken together, our results show that the novel trans-variant of HDV ribozyme can be used as a model for analyzing the process of HDV ribozyme self-cleavage.     

Kinetic Self-Cleavage Models for Permuted Variants of the Antigenomic HDV Ribozyme

The kinetic characteristics have been studied for noncircularly permuted variants of the human hepatitis delta virus antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Different ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic constant of the first reaction phase and the portion of the ribozyme molecules that self-cleaved during the first phase. Partial restoration of the initial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the data obtained, a scheme of the self-cleavage reaction has been proposed including: (i) activation of the ribozyme with energy of 40–50 kcal/mol and a characteristic time of several deciminutes under optimal reaction conditions; (ii) fast and reversible reaction of the phosphodiester bond cleavage; (iii) reaction leading to isomerization of the 3",5"-phosphodiester bond to the 2",5" bond in the self-cleavage site with a characteristic activation time of tens of minutes; and (iv) practically irreversible conformational change leading to fixation of the cleavage by immobilization of the 5"-terminal nucleotide of the product in the center of the formed structure and displacement of the 3"-terminal nucleotide to the periphery. The latter process has a characteristic time of tens of minutes and a low activation energy.     

Inhibition of Hepatitis Delta Virus Genomic Ribozyme Self-Cleavage by Aminoglycosides

Subgenomic regions of hepatitis delta virus (HDV) RNA contains ribozyme whose activities are important to viral life cycles and depend on a unique pseudoknot structure. To explore the characters of HDV ribozyme, antibiotics of the aminoglycoside, which has been shown inhibiting self-splicing of group I intron and useful in elucidating its structure, were tested for their effect on HDV genomic ribozyme. Aminoglycosides, including tobramycin, netromycin, neomycin and gentamicin effectively inhibited HDV genomic ribozyme self-cleavage in vitro at a concentration comparable to that inhibiting group I intron self-splicing. The extent of inhibition depended upon the concentration of magnesium ion. Chemical modification mapping of HDV ribozyme RNA indicated that the susceptibility of nucleotide 703 to the modifying agent was enhanced in the presence of tobramycin, suggesting a conformational shift of HDV ribozyme, probably due to an interaction with the aminoglycoside. Finally, we examined the effect of aminoglycoside on HDV cleavage and replication in cell lines, however, none of the aminoglycoside effective in vitro exerted suppressive effects in vivo. Our results represented as an initial effort in utilizing aminoglycoside to probe the structure of HDV ribozyme and to compare its reaction mechanism with those of other related ribozymes.     

双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用

为探讨双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用,观察双位点核酶对单一核酶体外剪切的增强作用,同时比较串联核酶和混合核酶的体外切割作用,构建了核酶Rz1,Rz3, Rz1和Rz3的串联核酶(Rz13)体外转录载体, 经体外转录后切割靶RNA. 结果表明:双位点核酶,无论是串联或混合核酶均可增强单一核酶的体外切割作用, 串联和混合核酶中的单一核酶可独立发挥作用;当串联和混合数目为2个时,两者的切割效率差别不大(P＞0.05).     

Addition of an Extra Substrate Binding Site and Partial Destabilization of Stem Structures in HDV Ribozyme Give Rise to High Sequence-Specificity for its Target RNA

Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.     

双价核酶对烟草花叶病毒的两个靶序列的专一切割作用

报道了两个最简单的多价核酶即双价核酶RZ34和RZl3的构建和体外作用情况。实验结果表明,这两个双价核酶能分别对两个不同的TMV底物或其混合物施行专一切割。双价核酶对单十底物的作用效率与其相应的单价核酶相似。还就双价核酶内的单元核酶的相对位置对其专一性和作用效率的影响进行了探索。     

丁型肝炎病毒核酶的结构特点与催化作用机制

丁型肝炎病毒(HDV)核酶是小核酶的一种,在分子结构和作用机制等方面都有许多不同于其它核酶的特性。以其晶体结构的揭示为基础,近几年对其立体构型及催化机制方面的研究取得了很大进展,尤其是发现HDV核酶的胞嘧啶侧链在生理条件下能发挥一般酸碱催化作用(generalacidbasecatalysis),引起了极大关注。对HDV核酶结构和催化机制的研究,将使核酶被有目的地改造,并极大地推动它在应用方面的研究。     

Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.     

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

丙型肝炎病毒RNA特异性核酶的切割活性研究

针对丙型肝炎病毒RNA(HCV-RNA)的5′非编码区和部分C区的二级结构,设计并合成了四个不同的锤头型核酶(ribozyme A, ribozyme B, ribozyme C₁, ribozyme C₂).首先应用体外切割实验筛选出作用于HCV-RNA起始密码子上游GTA↓位点的核酶RzA有较好的活性.为初步验证核酶RzA在细胞内的切割活性,经脂质体介导,将RzA-RNA与另一携带该核酶靶基因的质粒表达载体pCl-neo-luciferase(载体中荧光素酶基因受核酶靶基因的调控)共转染HepG₂细胞.通过测定荧光素酶基因的表达证实了核酶在细胞内有较好的切割活性.在此实验基础上,把RzA基因克隆至pCl-neo质粒表达载体中,再次经脂质体介导,将重组的表达载体pCl-neo-RzA与携带该核酶靶基因的质粒表达载体pCl-neo-luciferase共转染HepG₂细胞,获得了更好的切割效果.     

Interaction between RNA molecules of a two-component trans analog of antigenomic HDV ribozyme

A study was made of the association of the RNA components forming a B:LS two-component rans analog of the antigenomic HDV ribozyme. The B:LS ribozyme differed from known trans ribozymes in the sizes and nucleotide sequences of its components (33 and 34 nt, respectively), the topology of its functional parts, and the lack of a very short cleavage product. Compared to the cis ribozyme, B:LS showed similar dependences on the reaction conditions (Mg²⁺ concentration, pH, temperature) and a similar biphasic kinetic curve of self-cleavage. The kinetic model of B:LS self-cleavage (available at www.cardio.ru/labgen/RZ_e.html) describes a possible cause of the biphasic kinetic curve as a change in the rate-limiting step of consecutive conformational transitions accompanying self-cleavage. Another possible cause is an interaction between the molecules involved in cleavage, i.e., multimerization of whole ribozyme molecules with their components or the reaction products. B:LS provides a convenient model for studying such interactions, since the mode of component binding allows generation of 1B:2LS and 2B:1LS complexes as well as complexes with the cleavage products. Nondenaturing PAGE was used to study the factors affecting association and dissociation of the ribozyme components. The possibility of interactions between the RNA components of the cis and trans ribozymes was demonstrated experimentally. It was shown that the ribozyme is capable of multimerization when LS is in excess over B and that the cleavage products are not significantly involved in this process. The results suggest intermolecular interactions for the cleavage of the natural cis ribozyme.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

表达反义核酶RNA的转基因水稻对矮缩病毒复制和症状的抑制作用

用基因枪法将含有ＲＤＶ第五片段反义核酶序列基因的植物表达载体ｐＲＯＫＩＩ转化水稻幼胚,在Ｇ４１８存在的条件下,约２～３个月可筛选出抗性愈伤,转入分化培养基中培养可分化出幼苗。经Ｓｏｕｔｈｅｒｎ杂交法检测为阳性的水稻幼苗进行抗病性测定显示,转ＲＤＶ反义核酶基因的水稻植株对ＲＤＶ的复制和症状有显著抑制作用。转基因植株发病较轻,并能部分结实,而对照植株则明显矮化且大多不能抽穗。     

抗水稻矮缩病毒的反义核酶及复制酶部分序列的合成、克隆及其水稻表达载体的构建

采用反转录－聚合酶链式反应方法（ＲＴＰ－ＣＲ）,在人工合成的引物引导下,扩增出水稻矮缩病毒基因组第一片段（Ｓ１）全长序列及第五片段的部分序列（ＳＳⅢ）．将扩增的片段分别克隆到克隆载体ｐＧＥＭ７Ｚｆ（＋）及ｐＵＣ１９的ｓｍａｌ位点上,并进行了序列测定。在此基础上,利用ｐＣＲ引入的方法将核酶序列引入到Ｓ５Ⅲ片段反义链上以构成反义核酶基因Ｓ５ⅢＲ,将Ｓ１片段５＇端部分序列（Ｓ１－１）及Ｓ５ⅢＲ基因克隆到植物表达载体ｐＲＯＫⅡ上,构建成水稻转化载体ｐＲＯＫ－Ｓ１－１＇及ｐＲＯＫ－Ｓ５ⅢＲ。     

抗BmNPV即刻早期蛋白三联ribozyme在家蚕细胞中的表达

为了阻断家蚕核多角体病毒（BmNPV）的基因表达,以BmNPV的即刻早期蛋白基因(IE)为靶序列,设计了三联ribozyme．体外切割反应表明,该ribozyme能特异地切割靶序列的mRNA;细胞实验表明,细胞中表达的ribozyme也能够特异地切割靶序列,从而使受BmNPV感染的Bm-N细胞中的多角体减少约30％．     

Structural dynamics of precursor and product of the RNA enzyme from the hepatitis delta virus as revealed by molecular dynamics simulations

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme involved in the replication of a human pathogen, the hepatitis delta virus. Recent crystal structures of the precursor and product of self-cleavage, together with detailed kinetic analyses, have led to hypotheses on the catalytic strategies employed by the HDV ribozyme. We report molecular dynamics (MD) simulations (approximately 120 ns total simulation time) to test the plausibility that specific conformational rearrangements are involved in catalysis. Site-specific self-cleavage requires cytidine in position 75 (C75). A precursor simulation with unprotonated C75 reveals a rather weak dynamic binding of C75 in the catalytic pocket with spontaneous, transient formation of a H-bond between U-1(O2') and C75(N3). This H-bond would be required for C75 to act as the general base. Upon protonation in the precursor, C75H+ has a tendency to move towards its product location and establish a firm H-bonding network within the catalytic pocket. However, a C75H+(N3)-G1(O5') H-bond, which would be expected if C75 acted as a general acid catalyst, is not observed on the present simulation timescale. The adjacent loop L3 is relatively dynamic and may serve as a flexible structural element, possibly gated by the closing U20.G25 base-pair, to facilitate a conformational switch induced by a protonated C75H+. L3 also controls the electrostatic environment of the catalytic core, which in turn may modulate C75 base strength and metal ion binding. We find that a distant RNA tertiary interaction involving a protonated cytidine (C41) becomes unstable when left unprotonated, leading to disruptive conformational rearrangements adjacent to the catalytic core. A Na ion temporarily compensates for the loss of the protonated hydrogen bond, which is strikingly consistent with the experimentally observed synergy between low pH and high Na+ concentrations in mediating residual self-cleavage of the HDV ribozyme in the absence of divalents.