A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.     

猪瘟病毒糖蛋白E^rns中和表位的鉴定和比较

猪瘟病毒 (CSFV)囊膜结构糖蛋白Erns(gp4 8)是诱导机体产生中和抗体及激发保护性免疫应答的第二抗原蛋白。E2和Erns与细胞表面受体的相互作用介导CSFV感染细胞的过程。Erns具有RNA酶活性 ,影响病毒自身复制并涉及对病毒的中和效应。采用抗CSFValfortT櫣ｂｉｎｇｅｎ毒株Ｅｒｎｓ糖蛋白的 1B5 ,b4_2 2和 2 4 16单克隆中和抗体 ,筛选噬菌体展示的 12肽随机肽库 ,进行Erns中和表位的鉴定和比较 ,获得分别针对 1B5、b4_2 2和 2 4 16单克隆抗体的 3个主要中和表 (拟 )位基序WxNxxP、DKNR (Q)G和A(T)CxYxKN ,分别定位于Erns的 35 1位～ 35 6位或 348位～ 35 0位、384位～ 386及 32 2位～ 32 3位、380位～ 386位氨基酸区域。分析表 (拟 )位基序与单克隆抗体的免疫反应性差异。b4_2 2和 2 4 16单克隆抗体识别基序存在共有序列KN ,识别Erns中的相似抗原区 ,但其侧翼序列及免疫印迹、免疫荧光抗体抑制试验结果均存在显著差异     

A Streptomyces coelicolor functional orthologue of Escherichia coli RNase E shows shuffling of catalytic and PNPase-binding domains

Previous work has detected an RNase E-like endoribonucleolytic activity in cell extracts obtained from Streptomyces. Here, we identify a Streptomyces coelicolor gene, rns, encoding a 140 kDa protein (RNase ES) that shows endoribonucleolytic cleavage specificity characteristic of RNase E, confers viability on and allows propagation of Escherichia coli cells lacking RNase E and accomplishes RNase E-like regulation of plasmid copy number in E. coli. However, notwithstanding its complementation of rne-deleted E. coli, RNase ES did not accurately process 9S rRNA from E. coli. Additionally, whereas RNase E is normally required for E. coli survival, rns is not an essential gene in S. coelicolor. Deletion analysis mapped the catalytic domain of RNase ES near its centre and showed that regions located near the RNase ES termini interact with an S. coelicolor homologue of polynucleotide phosphorylase (PNPase) - a major component of E. coli RNase E-based degradosomes. The interacting arginine- and proline-rich segments resemble the C-terminally located degradosome scaffold region of E. coli RNase E. Our results indicate that RNase ES is a structurally shuffled RNase E homologue showing evolutionary conservation of functional RNase E-like enzymatic activity, and suggest the existence of degradosome-like complexes in Gram-positive bacteria.     

Expression and functional characterization of classical swine fever virus E(rns) protein

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with unusual RNase activity. Recently, E(rns) was found to have a new function of counteracting the beta-interferon (IFN-beta) induction pathway. In this study, wildtype ErnsSM and two mutated E(rns) proteins ErnsH297k and ErnsH346k were expressed in insect cells and purified for RNase activity and function analysis. RNase activity assay in vitro demonstrated that only wildtype E(rns) protein had RNase activity. However, both wildtype ErnsSM and the two mutated E(rns)ErnsH297k and ErnsH346k as exogenous proteins had a block effect on Newcastle disease virus (NDV)-mediated IFN-beta promoter induction.     

A novel role of classical swine fever virus E(rns) glycoprotein in counteracting the newcastle disease virus (NDV)-mediated IFN-beta Induction

E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that E(rns) counteracts Newcastle disease virus (NDV)-mediated induction of IFN-beta. For this purpose, E(rns) fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, E(rns)-EGFP was found to prevent IFN-beta promoter-driven luciferase expression and block the induction of IFN-beta promoter mediated by NDV in a dosedependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-beta mRNA in NDV-infected PK15 cells was observed in the presence of E(rns)-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, E(rns)-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-beta. These evidences establish a novel function for CSFV E(rns) glycoprotein in counteraction of the IFN-beta induction pathway.     

Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines

An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein E(rns) of classical swine fever virus (CSFV) was used to rescue CSFV E(rns) deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of E(rns) from this cell line were indistinguishable from those of CSFV E(rns). Two E(rns) deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of E(rns) (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of E(rns) antibodies, due to a deletion of 66 amino acids in E(rns). The E(rns) deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by E(rns)-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that E(rns) from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) E(rns) in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.     

Association and dissociation kinetics of colicin E3 and immunity protein 3: convergence of theory and experiment

The rapid binding of cytotoxic colicin E3 by its cognate immunity protein Im3 is essential in safeguarding the producing cell. The X-ray structure of the E3/Im3 complex shows that the Im3 molecule interfaces with both the C-terminal ribonuclease (RNase) domain and the N-terminal translocation domain of E3. The association and dissociation rates of the RNase domain and Im3 show drastically different sensitivities to ionic strength, as previously rationalized for electrostatically enhanced diffusion-limited protein-protein associations. Relative to binding to the RNase domain, binding to full-length E3 shows a comparable association rate but a significantly lower dissociation rate. This outcome is just what was anticipated by a theory for the binding of two linked domains to a protein. The E3/Im3 system thus provides a powerful paradigm for the interplay of theory and experiment.     

Identification of antigenic regions of the Erns protein for pig antibodies elicited during classical swine fever virus infection

The structural glycoprotein E(rns) of classical swine fever virus (CSFV) is one of the major antibody targets upon infection of pigs with the virus. Molecular dissection of the structure of E(rns) would define the minimal immunodominant regions that induce antibody responses after infection and may thus help design an effective diagnostic reagent or vaccine. In this study, deletion analysis was made within amino acids (aa) 297 to 776 of the CSFV Alfort/187 polyprotein containing the large C-terminal portion of the E(rns) protein (aa 27 to 227), the entire E1 protein (aa 1 to 195), and the N-terminal portion of the E2 protein (aa 1 to 87). Various protein fragments with target deletions from N- or/and C-terminal ends were constructed with pET30, expressed in Escherichia coli and probed on Western blots with antisera from pigs infected with CSFV. This has resulted in the identification within E(rns) of three overlapping antigenic regions: AR1(E(rns)aa 65-145), AR2 (E(rns)aa 84-160) and AR3 (E(rns)aa 109-220). N- or C-terminal deletions as small as 3 residues introduced into these regions disrupt their reactivity with antibodies, indicating that they are the minimum requirements for recognition by pig antibodies. The three minimal antigenic regions correlated well with the hydropathy profiles and the 3D structural model of E(rns). Each individual region and a protein fragment containing AR1, AR2 and AR3 reacted equally well with pig anti-CSFV sera. Since variable and conserved sequences are present within the three overlapping antigenic regions of E(rns) of different pestiviruses, specific serological detection of CSFV infection or broad detection of pestivirus infections may be achieved with the use of a single E(rns) region or a combination of two or three E(rns) regions.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

The carboxy-terminal sequence of the pestivirus glycoprotein E(rns) represents an unusual type of membrane anchor

The E(rns) protein is a structural glycoprotein of pestiviruses that lacks a typical membrane anchor sequence and is known to be secreted from the infected cell. However, major amounts of the protein are retained within the cell and attached to the virion by a so far unknown mechanism. Transient-expression studies with cDNA constructs showed that in a steady-state situation, 16% of the protein is found in the supernatant of the transfected cells while 84% appears as intracellular protein. We show here that E(rns) represents a membrane-bound protein. Membrane binding occurs via the carboxy-terminal region of E(rns). By fusion of this sequence to the carboxy terminus of green fluorescent protein (GFP), the subcellular localization of the reporter protein switched from cytosolic to membrane bound. A core sequence of 11 amino acids necessary for membrane binding was elicited in truncation experiments with GFP constructs. However, this peptide is not sufficient to confer membrane anchoring but needs either upstream or downstream accessory sequences. Analyses with different extraction procedures showed that E(rns) is neither easily stripped from the membrane, like a peripheral membrane protein, nor as tightly membrane bound as a transmembrane protein.     

The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain

The SCO2299 gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N-terminal region shows high amino acid sequence similarity to RNase HI, whereas its C-terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature-sensitive growth defect of an Escherichia coli RNase H-deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA.DNA hybrid in vitro. The N-terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C-terminal domain showed no CobC-like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N-terminal and C-terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.     

Role of N-glycan trimming in the folding and secretion of the pestivirus protein E(rns)

N-glycosylation inhibitors have antiviral effect against bovine viral diarrhea virus. This effect is associated with inhibition of the productive folding pathway of E1 and E2 envelope glycoproteins. E(rns) is the third pestivirus envelope protein, essential for virus infectivity. The protein is heavily glycosylated, its N-linked glycans counting for half of the apparent molecular weight. In this report we address the importance of N-glycan trimming in the biosynthesis, folding, and intracellular trafficking of E(rns). We show that E(rns) folding is not assisted by calnexin and calreticulin; however, the protein strongly interacts with BiP. Consistently, the N-glycan trimming is not a prerequisite for either the acquirement of the E(rns) native conformation, as it retains the RNase enzymatic activity in the presence of alpha-glucosidase inhibitors, or for dimerization. However, E(rns) secretion into the medium is severely impaired suggesting a role for N-glycosylation in the transport of the glycoprotein through the secretory pathway.     

C-terminal WxL domain mediates cell wall binding in Enterococcus faecalis and other gram-positive bacteria

Analysis of the genome sequence of Enterococcus faecalis clinical isolate V583 revealed novel genes encoding surface proteins. Twenty-seven of these proteins, annotated as having unknown functions, possess a putative N-terminal signal peptide and a conserved C-terminal region characterized by a novel conserved domain designated WxL. Proteins having similar characteristics were also detected in other low-G+C-content gram-positive bacteria. We hypothesized that the WxL region might be a determinant of bacterial cell location. This hypothesis was tested by generating protein fusions between the C-terminal regions of two WxL proteins in E. faecalis and a nuclease reporter protein. We demonstrated that the C-terminal regions of both proteins conferred a cell surface localization to the reporter fusions in E. faecalis. This localization was eliminated by introducing specific deletions into the domains. Interestingly, exogenously added protein fusions displayed binding to whole cells of various gram-positive bacteria. We also showed that the peptidoglycan was a binding ligand for WxL domain attachment to the cell surface and that neither proteins nor carbohydrates were necessary for binding. Based on our findings, we propose that the WxL region is a novel cell wall binding domain in E. faecalis and other gram-positive bacteria.     

Identification and characterization of rns4/vps32 mutation in the RNase T1 expression-sensitive strain of Saccharomyces cerevisiae: Evidence for altered ambient response resulting in transportation of the secretory protein to vacuoles

We previously reported a genetic analysis of the growth-inhibitory effect caused by the overexpression of the Aspergillus oryzae rntA gene, encoding RNase T1 (Ribonuclease T1), in Saccharomyces cerevisiae. Subsequently, rns (ribonuclease T1 sensitive) mutants with mutations in the rns1 (DSL1), rns2 (UMP1), and rns3 (SEC17) genes, were identified. In the present study, rns4 (VPS32/SNF7) gene mutation was identified by complementation of tunicamycin sensitivity. While the rns4 mutant exhibited sensitivity to ambient stress conditions (200 mM CaCl(2), 1M NaCl and pH 8.0), genome-wide expression analysis revealed a similar pattern of genes up-regulated as was observed under nitrogen depletion condition by Gasch et al. [Mol. Biol. Cell 11 (2000) 4241]. Notably, the genes participating in autophagy (ATG4 and ATG8), the genes encoding a vacuolar protease (PRB1), vacuolar protease inhibitors (PAI3, PBI2 and TFS1) and YHR138c (a PBI2 homolog) were up-regulated in the rns4 mutant. Interestingly, the RNase T1*-GFP fusion protein (*inactive form) expressed in the rns4 mutant strain localized at the ER and vacuole under both stress or no-stress conditions. In contrast, the RNase T1*-GFP fusion protein expressed in the wild-type strain could not be detected under no-stress conditions, however, a stress-dependent localization of the fusion protein was observed at the vacuole. Since, the rns4 mutant exhibited a partial starvation-like response in spite of a rich ambient environment, leading to transportation of the secretory protein to the vacuole and accumulation in the endoplasmic reticulum, the present findings implicate a novel role for Rns4/Vps32 in proper response and adaptation to ambient conditions.     

Functional characterization of a cellulose binding xylanase from Fusarium oxysporum

Summary An extracellular endoxylanase from Fusarium oxysporum binds onto crystalline cellulose. A small peptide (~ 2kDa) could be isolated after partial proteolysis of the native protein. It consists of 18 amino acids, is located in the C-terminal region of the protein and corresponds functionally to a cellulose binding domain (CBD), the first one to be reported in a fungal xylanase. The amino acid sequence of this peptide shows no homology with any known CBD.     

Characterization of the arabidopsis formin-like protein AFH1 and its interacting protein

A partial cDNA encoding an Arabidopsis thaliana FH (Formin Homology) protein (AFH1) was used as a probe to clone a full length AFH1 cDNA. The deduced protein encoded by the cDNA contains a FH1 domain rich in proline residues and a C-terminal FH2 domain which is highly conserved amongst FH proteins. In contrast to FH proteins of other organisms, the predicted AFH1 protein also contains a putative signal peptide and a transmembrane domain suggesting its association with membrane. Cell fractionation by differential centrifugation demonstrated the presence of AFH1 in the Triton X-100 insoluble microsomal fraction. An Arabidopsis cDNA library was screened to identify proteins that interact with the C-terminal region of AFH1 using yeast two-hybrid assays, and one of the isolated cDNAs encoded a novel protein, FIP2. Experiments using recombinant proteins expressed in E. coli demonstrated that FIP2 interacted directly with AFH1. The amino acid sequence of FIP2 has partial homology to bacterial putative membrane proteins and animal A-type K+ ATPases. AFH1 may form a membrane anchored complex with FIP2, which might be involved in the organization of the actin cytoskeleton.     

Identification of domains of the human papillomavirus type 11 E1 helicase involved in oligomerization and binding to the viral origin

The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by single-stranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.