Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn²⁺) are preferred rather than magnesium ions (Mg²⁺), whereas zinc ions (Zn²⁺) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.     

Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants

Turnip crinkle virus (TCV) is a small, plus-sense, single-stranded RNA virus of plants. A virus-coded protein, p88, which is required for replication has been expressed and purified from Escherichia coli. In vitro assays revealed that the recombinant p88 has an RNA-dependent RNA polymerase (RdRp) activity and can also bind to RNA. Deletion of the N-terminal region in p88 resulted in a more active RdRp, while further deletions abolished RdRp activity. Comparison of the E. coli-expressed p88, the N-terminal deletion mutant of p88, and a TCV RdRp preparation obtained from infected plants revealed that these preparations show remarkable similarities in RNA template recognition and usage. Both the recombinant and the plant TCV RdRp preparations are capable of de novo initiation on both plus- and minus-strand satC and satD templates, which are small parasitic RNAs associated with TCV infections. In addition, these RdRp preparations can efficiently recognize the related Tomato bushy stunt virus promoter sequences, including the minus- and plus-strand initiation promoters. Heterologous viral and artificial promoters are recognized poorly by the recombinant and the plant TCV RdRps. Further comparison of the single-component recombinant TCV RdRp and the multicomponent plant TCV RdRp will help dissect the functions of various components of the TCV replicase.     

RNA-Dependent RNA Polymerase in Nuclei of Cells Infected with Influenza Virus

Nuclei purified from chicken embryo fibroblast cells infected with influenza (fowl plague) virus contain an RNA-dependent RNA polymerase. The in vitro activity of this enzyme is insensitive to actinomycin D, and is completely destroyed by preincubation with ribonuclease. Enzyme induction is prevented if cells are treated with actinomycin D or cycloheximide at the time of infection. RNA-dependent RNA polymerase activity increases rapidly in cell nuclei from 1 h postinfection, reaches a maximum at 3 to 4 h, then declines; a similar RNA polymerase activity in the microsomal cell fraction increases from 2 h postinfection and reaches a maximum at 5 to 6 h. The characteristics of the nuclear and microsomal enzymes in vitro are similar with respect to pH and divalent cation requirements. The in vitro products of enzyme activity present in the nuclear and microsomal fractions of cells infected for 3 and 5 h were characterized by sucrose density gradient analysis, and annealing to virion RNA. The microsomal RNA polymerase product contained 67 and 93% RNA complementary to virion RNA at 3 and 5 h, respectively; for the nuclear RNA polymerase product these values were 40% in each case.     

New Non-nucleoside Inhibitors of Hepatitis C Virus RNA-Dependent RNA Polymerase

Recombinant RNA-dependent RNA polymerase of hepatitis C virus was purified using a bacterial expression system (Escherichia coli). The system for enzyme activity detection was optimized. The maximum activity was achieved when the reaction was carried out at 30 degrees C in the presence of 3 mM Mg2+ or 0.75 mM Mn2+. Among alpha- and beta-pyrogallaldehydes, effective inhibitors were found. It was shown that they acted at the primer elongation stage, and their binding to the protein is reversible.     

Functions of Conserved Motifs in the RNA-Dependent RNA Polymerase of a Yeast Double-Stranded RNA Virus

At least eight conserved motifs are visible in the totivirus RNA-dependent RNA polymerase (RDRP). We have systematically altered each of these in the Saccharomyces cerevisiae double-stranded RNA virus ScVL1 by substituting the conserved motifs from a giardiavirus. The results help define the conserved regions of the RDRP involved in polymerase function and those essential for other reasons.     

A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3'-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5' end or 45 nt from the 3' end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3'-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3' end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3' end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3' end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.     

Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the template-dependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science.Several model systems for biochemical study of viral RNA-dependent RNA synthesis exist (4, 19, 20, 25, 37, 42). Well-characterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47-49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60).RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed conformation, even in the absence of template, in contrast to DNA-dependent RNA polymerases, which transition from open to closed complexes upon template recognition (13). The closed form of the phage φ6 RdRp has been proposed to allow specific recognition of the single-stranded viral RNA (7). The template channel formed by the closed structure, however, is too narrow to accommodate the partially duplexed RNA that forms during RNA synthesis, and hence, the closed conformation needs to undergo significant rearrangements in the ternary complex. Biswal et al. (3) have captured an X-ray crystallographic structure of a partially open conformation of the HCV RdRp. Bovine viral diarrhea virus (BVDV) RdRp was also shown to exist in a partially open conformation (11). Ranjith-Kumar and Kao (49) demonstrated that the HCV RdRp could initiate RNA synthesis from a circular RNA template, and thus, the threading of a single-stranded RNA into the template channel is not required for de novo-initiated RNA synthesis. Altogether, these results raise the possibility that the HCV RdRp can undergo rearrangements from the closed conformation seen in the crystal structure prior to de novo initiation.A secondary structure that extends from the finger to the thumb subdomains, named the Δ1 loop, has been proposed to serve as a gate to cover the template channel and regulate the switch from de novo initiation to elongation (5, 10). Mutations that affect the interaction between the Δ1 loop and the mostly hydrophobic residues that it contacts have resulted in polymerases that are defective for de novo initiation but can bind to partially duplexed RNA and can extend from the 3′ terminus of an RNA primer (10).Two general models for RNA synthesis by the HCV RdRp can be proposed (Fig. ​(Fig.1).1). The first posits that the HCV RdRp functions as a monomer at least during de novo initiation because the closed template channel is needed for specific recognition of the template (5, 7, 10). It was presumed that the Δ1 loop and thumb domain interaction in the HCV RdRp is stable and mutations that disrupted this interaction would render the enzyme catalytically inactive (5, 24). However, a deletion of five residues in the tip of the Δ1 loop did not prevent RNA synthesis from a primed template by the polymerase (10). Furthermore, a genotype 2a RdRp was crystallized in a form with altered interaction between the Δ1 loop and thumb domain in comparison to the 1b RdRp (3). Interestingly, a low-affinity GTP binding site exists on the thumb domain close to the base of the Δ1 loop binding pocket. GTP binding at this site has been proposed to stabilize the Δ1 loop and thumb domain interactions, favoring the closed monomer model (6). A second model is based on the reports that HCV RdRp can oligomerize and that oligomerization increases its activity (12, 16, 46, 54). The dimer could be active due to either the second subunit increasing the stability of the Δ1 loop and thumb interactions in the first subunit to increase de novo initiation or the two subunits forming a common template-binding domain (Fig. ​(Fig.1).1). Here we have attempted to determine whether monomers or oligomers of the HCV RdRp can better perform de novo initiation using biochemical and biophysical analyses.Open in a separate windowFIG. 1.Models for RNA synthesis by the HCV RdRp. The monomer model is based on the central tenet that intramolecular interactions within an RdRp molecule regulate the modes of RNA synthesis. The curved arrow represents the possible orientation of the template RNA. The oligomer model is an adaptation from the dimer model of the norovirus RdRp (18). T, P, and F represent the thumb, palm, and finger domains, respectively, in different shades of gray, and the thick black line connecting the thumb and finger domains represents the Δ1 loop.     

兔出血症病毒衣壳蛋白基因在毕赤酵母中的表达

将RHDVVP60基因插入酵母转移载体pPICZB中转化毕赤酵母菌GS115株 ,经筛选获得染色体基因组中整合入VP60基因的重组酵母菌。以甲醇诱导培养后经SDS-PAGE和Westernblot检测表达产物 ,在60kD处出现一特异蛋白条带 ,表明RHDV的衣壳蛋白得到了成功表达。血凝试验表明 ,表达的重组蛋白具有血凝特性 ,可以凝集人“O”型红细胞 ,血凝价达 2.8,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组酵母表达的衣壳蛋白可以在酵母菌体内自聚成大小约4.0nm ,和天然RHDV病毒子在物理形态上类似的病毒样颗粒 (VLPs)。该病毒样颗粒与兔抗RHDV高免血清作用后可被凝集成团 ,表明该VLPs与天然RHDV在抗原性上也极为相似。     

兔出血症病毒与细小病毒抗原相关性试验

用间接ELISA、ELISA交叉阻断法和交叉血凝抑制试验对兔出血症病毒(RHDV)与6种细小病毒进行抗原相关性试验。用间接ELISA证实,RFIDV与它们有轻度交叉关系,其抗原相关值分别为:小鼠细小病毒(MVM)5.59％;鹅细小病毒(GPV)3.54％;猪细小病毒(PPV)1.76％;水貂肠炎病毒0.7％。细小病毒间的抗原相关值:MEV与PPV为31.6％,MEV与MVM为35.36％;而CPV与MEM、PPV、MVM的相关值均为零,即无相关性。在ELISA交叉阻断法中证实:犬细小病毒(CPV)、猫泛白细胞减少症病毒(FPV)和MEV均不能阻断RHDV与其抗体结合,仅GPV有轻度阻断作用,其最大阻断率为40％。在血凝交叉抑制试验中,未发现RHDV与细小病毒及其相应抗体间存在交叉抑制现象。以上结果表明RHDV与细小病毒在血清学方面有轻度相关性。     

兔出血症病毒结构多肽分析

粗提病毒经Ｓｅｐｈａｒｏｓｅ４Ｂ层析后,获得纯化的兔出血症病毒（ＲＨＤＶ）。提纯病毒经过ＳＤＳ－ＰＡＧＥ经考马斯亮蓝染色显示Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ７条多肽,凝胶扫描显示Ａ为ＲＨＤＶ主要结构多肽。用多抗和单抗作免疫转印分析,证实Ａ、Ｂ、Ｃ、Ｄ、Ｅ、和Ｇ为结构多肽,此６条结构多肽间的抗原关系十分密切。     

兔出血症细小病毒血清学的研究

应用免疫双扩散和酶联免疫吸附分析(ELISA)对七个不同地区兔出血症病原分离物进行了血清学比较研究。本试验用免疫双扩散和ELISA,证明七个兔出血症病毒分离物之间存在着基本相同的血清学关系,其中A_2R—3和H.E.二株抗原之间存在较大差异.证实我国流行的兔出血症病毒属于一个血清型,但不同地区的分离株可能出现亚型。