共查询到20条相似文献,搜索用时 15 毫秒
1.
Guardado-Calvo P Vazquez-Iglesias L Martinez-Costas J Llamas-Saiz AL Schoehn G Fox GC Hermo-Parrado XL Benavente J van Raaij MJ 《Journal of virology》2008,82(22):11208-11216
Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural σA protein is a major component of the inner capsid, clamping together λA building blocks. σA has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed σA by molecular replacement and refined it using data to 2.3-Å resolution. Twelve σA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of σA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of σA to double-stranded RNA. The minimal length of double-stranded RNA required for σA binding was observed to be 14 to 18 bp. 相似文献
2.
Avian reovirus temperature-sensitive mutant tsA12 has a lesion in major core protein sigmaA and is defective in assembly 下载免费PDF全文
Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperature-sensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced approximately 100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced approximately 100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein sigmaA. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the sigmaA protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu(158) substitution in ARV sigmaA lies directly under the outer face of the sigmaA protein. This may cause a perturbation in sigmaA such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly. 相似文献
3.
Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein 总被引:1,自引:0,他引:1
Yamashita S Nagata T Kawazoe M Takemoto C Kigawa T Güntert P Kobayashi N Terada T Shirouzu M Wakiyama M Muto Y Yokoyama S 《Protein science : a publication of the Protein Society》2011,20(1):118-130
The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue. 相似文献
4.
Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection. 总被引:15,自引:3,他引:15 下载免费PDF全文
We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death. 相似文献
5.
6.
Research on immune function in evolutionary ecology has frequently focused on avian ectoparasites (e.g., mites and lice). However, host immunogenetics involved with bird resistance to ectoparasites has not been determined. The critical role of the major histocompatibility complex (MHC) in adaptive immunity and high genetic variation found within the MHC make this gene complex useful for exploring the immunogenetic basis for bird resistance to ectoparasites. The objective of this study was to determine if the avian MHC influenced resistance to a blood-feeding ectoparasite. Four congenic lines of chickens, differing only at the MHC, were comparatively infested with a cosmopolitan ectoparasite of birds-northern fowl mite (NFM)-which is also a serious pest species of poultry. Mite infestations were monitored over time and mite densities (weekly and maximum) were compared among lines. Chickens with the MHC haplotype B21 were relatively resistant to NFM, compared with birds in the B15 congenic line (P < 0.02). To test for similar effects in an outbred genetic background, a separate experiment was performed with 107 commercial chickens (white leghorn, W-36 strain) infested with NFM. Hens were genotyped using a MHC microsatellite marker (LEI0258) and associations between MHC haplotype and NFM density were tested. The highest peak NFM populations occurred more often on hens with the B15 haplotype versus the B21 haplotype (P = 0.012), which supported the results of the congenic study. These data indicate the avian MHC influences ectoparasite resistance, which is relevant to disease ecology and avian-ectoparasite interaction. 相似文献
7.
8.
9.
10.
Proteolytic cleavage of the reovirus sigma 3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region. 下载免费PDF全文
The reovirus capsid protein sigma 3 was examined for double-stranded RNA (dsRNA)-binding activity by Northwestern (RNA-protein) blot analysis. Treatment of virion-derived sigma 3 protein with Staphylococcus aureus V8 protease led to an increase in the dsRNA-binding activity associated with the C-terminal fragment of the protein. Recombinant C-terminal fragments of the sigma 3 protein were expressed in Escherichia coli from the S4 cDNA of reovirus serotype 1. These truncated sigma 3 proteins displayed proteolytic processing and dsRNA-binding activity similar to those observed for native, virion-derived sigma 3 protein as measured by Northwestern blot analysis. Construction of a modified pET3c vector, pET3Exo, allowed the production of 3'-terminal deletions of the S4 cDNA by using exonuclease III and rapid screening of the induced truncated sigma 3 proteins. An 85-amino-acid domain within the C-terminal portion of the sigma 3 protein which was responsible for dsRNA-binding activity was identified. The 85-amino-acid domain possessed a repeated basic amino acid motif which was conserved in all three serotypes of reovirus. Deletion of one of the basic motifs, predicted to be an amphipathic alpha-helix, destroyed dsRNA-binding activity. 相似文献
11.
Several zinc finger proteins have been discovered recently that bind specifically to double-stranded RNA. These include the mammalian JAZ and wig proteins, and the seven-zinc finger protein ZFa from Xenopus laevis. We have determined the solution structure of a 127 residue fragment of ZFa, which consists of two zinc finger domains connected by a linker that remains unstructured in the free protein in solution. The first zinc finger consists of a three-stranded beta-sheet and three helices, while the second finger contains only a two-stranded sheet and two helices. The common structures of the core regions of the two fingers are superimposable. Each finger has a highly electropositive surface that maps to a helix-kink-helix motif. There is no evidence for interactions between the two fingers, consistent with the length (24 residues) and unstructured nature of the intervening linker. Comparison with a number of other proteins shows similarities in the topology and arrangement of secondary structure elements with canonical DNA-binding zinc fingers, with protein interaction motifs such as FOG zinc fingers, and with other DNA-binding and RNA-binding proteins that do not contain zinc. However, in none of these cases does the alignment of these structures with the ZFa zinc fingers produce a consistent picture of a plausible RNA-binding interface. We conclude that the ZFa zinc fingers represent a new motif for the binding of double-stranded RNA. 相似文献
12.
The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs. 相似文献
13.
ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine (A-to-I). ADAR2 contains two tandem double-stranded RNA-binding motifs (dsRBMs) that are not only important for efficient editing of RNA substrates but also necessary for localizing ADAR2 to nucleoli. The sequence and structural similarity of these motifs have raised questions regarding the role(s) that each dsRBM plays in ADAR2 function. Here, we demonstrate that the dsRBMs of ADAR2 differ in both their ability to modulate subnuclear localization as well as to promote site-selective A-to-I conversion. Surprisingly, dsRBM1 contributes to editing activity in a substrate-dependent manner, indicating that dsRBMs recognize distinct structural determinants in each RNA substrate. Although dsRBM2 is essential for the editing of all substrates examined, a point mutation in this motif affects editing for only a subset of RNAs, suggesting that dsRBM2 uses unique sets of amino acid(s) for functional interactions with different RNA targets. The dsRBMs of ADAR2 are interchangeable for subnuclear targeting, yet such motif alterations do not support site-selective editing, indicating that the unique binding preferences of each dsRBM differentially contribute to their pleiotropic function. 相似文献
14.
Cell-type-specific repression of internal ribosome entry site activity by double-stranded RNA-binding protein 76 下载免费PDF全文
Translation of picornavirus plus-strand RNA genomes occurs via internal ribosomal entry at highly structured 5' untranslated regions. In addition to canonical translation factors, translation rate is likely influenced by supplementary host and viral trans-acting factors. We previously reported that insertion of a heterologous human rhinovirus type 2 internal ribosomal entry site (IRES) into the poliovirus (PV) genome, generating the chimeric virus PV-RIPO, selectively abrogates viral translation and propagation in neurons, which eliminate poliovirus's signature neuropathogenicity. While severely deficient in cells of neuronal lineage, the rhinovirus IRES promotes efficient propagation of PV-RIPO in cancer cells. Tumor-specific IRES function can be therapeutically exploited to direct viral cytotoxicity to cancer cells. Neuron-glioma heterokaryon analysis implicates neuronal trans-dominant inhibition in this effect, suggesting that host trans-acting factors repress IRES function in a cell-type-specific manner. We identified a set of proteins from neuronal cells with affinity for the rhinovirus IRES, including double-stranded RNA-binding protein 76 (DRBP76). DRBP76 associates with the IRES in neuronal but not in malignant glioma cells. Moreover, DRBP76 depletion in neuronal cells enhances rhinovirus IRES-driven translation and virus propagation. Our observations suggest that cell-type-specific association of DRBP76 with the rhinovirus IRES represses PV-RIPO translation and propagation in neuronal cells. 相似文献
15.
Mechanism of interferon action: functionally distinct RNA-binding and catalytic domains in the interferon-inducible, double-stranded RNA-specific adenosine deaminase. 总被引:6,自引:0,他引:6 下载免费PDF全文
The 1,226-amino-acid sequence of the interferon-inducible double-stranded RNA-specific adenosine deaminase (dsRAD) contains three copies (RI, RII, and RIII) of the highly conserved subdomain R motif commonly found in double-stranded RNA-binding proteins. We have examined the effects of equivalent site-directed mutations in each of the three R-motif copies of dsRAD on RNA-binding activity and adenosine deaminase enzyme activity. Mutations of the R motifs were analyzed alone as single mutants and in combination with each other. The results suggest that the RIII copy is the most important of the three R motifs for dsRAD activity and that the RII copy is the least important. The RIII mutant lacked detectable enzymatic activity and displayed greatly diminished RNA-binding activity. Site-directed mutations within the highly conserved CHAE sequence of the postulated C-terminal deaminase catalytic domain destroyed enzymatic activity but did not affect RNA-binding activity. These results indicate that the three copies of the RNA-binding R subdomain are likely functionally distinct from each other and also from the catalytic domain of dsRAD. 相似文献
16.
17.
RNA-binding proteins can strongly regulate and influence the cellular function and fate of an RNA molecule. Of the many described nucleic acid-binding domains, the double-stranded RNA-binding domain (dsRBD) is a highly specialized example found in a wide variety of proteins with diverse cellular functions. Mostly present in multiple copies and highly homologous to one another, the individual functional specificity of dsRBDs is now becoming apparent. Here we review recent evidence showing that single dsRBDs within individual proteins are capable of distinct in vivo functions. Not only does this enable dsRBD-containing proteins to increase their functional diversity but it also reveals novel and unexpected roles that dsRBDs can perform. 相似文献
18.
Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish. 相似文献
19.
Rotavirus RNA polymerase requires the core shell protein to synthesize the double-stranded RNA genome. 总被引:5,自引:6,他引:5 下载免费PDF全文
Rotavirus cores contain the double-stranded RNA (dsRNA) genome, RNA polymerase VP1, and guanylyltransferase VP3 and are enclosed within a lattice formed by the RNA-binding protein VP2. Analysis of baculovirus-expressed core-like particles (CLPs) has shown that VP1 and VP2 assemble into the simplest core-like structures with replicase activity and that VP1, but not VP3, is essential for replicase activity. To further define the role of VP1 and VP2 in the synthesis of dsRNA from viral mRNA, recombinant baculoviruses containing gene 1 (rBVg1) and gene 2 (rBVg2) of SA11 rotavirus were generated and used to express recombinant VP1 (rVP1) and rVP2, respectively. After purification, the proteins were assayed individually and together for the ability to catalyze the synthesis of dsRNA in a cell-free replication system. The results showed that dsRNA was synthesized only in assays containing rVP1 and rVP2, thus establishing that both proteins are essential for replicase activity. Even in assays containing a primer-linked mRNA template, neither rVP1 nor rVP2 alone directed RNA synthesis. Characterization of the cis-acting replication signals in mRNA recognized by the replicase of rVP1 and rVP2 showed that they were the same as those recognized by the replicase of virion-derived cores, thus excluding a role for VP3 in recognition of the mRNA template by the replicase. Analysis of RNA-protein interactions indicated that the mRNA template binds strongly to VP2 in replicase assays but that the majority of the dsRNA product neither is packaged nor stably associates with VP2. The results of replicase assays performed with mutant VP2 containing a deletion in its RNA-binding domain suggests that the essential role for VP2 in replication is linked to the protein's ability to bind the mRNA template for minus-strand synthesis. 相似文献
20.
RIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. Dicer requires double-stranded RNA-binding protein partners, such as PACT, for optimal activity. Here we show that PACT physically binds to the C-terminal repression domain of RIG-I and potently stimulates RIG-I-induced type I interferon production. PACT potentiates the activation of RIG-I by poly(I:C) of intermediate length. PACT also cooperates with RIG-I to sustain the activation of antiviral defense. Depletion of PACT substantially attenuates viral induction of interferons. The activation of RIG-I by PACT does not require double-stranded RNA-dependent protein kinase or Dicer, but is mediated by a direct interaction that leads to stimulation of its ATPase activity. Our findings reveal PACT as an important component in initiating and sustaining the RIG-I-dependent antiviral response. 相似文献