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1.
The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters. 总被引:1,自引:0,他引:1
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The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter. 相似文献
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A van Belkum B Cornelissen H Linthorst J Bol C Pley L Bosch 《Nucleic acids research》1987,15(7):2837-2850
The 3' terminal forty nucleotides of tobraviral RNAs readily fold into a tertiary structure, resembling that of tymo- and tobamoviral RNAs. The latter RNAs possess a tRNA-like structure at their 3' end that is recognized by a number of tRNA-specific enzymes (Rietveld et al. (1984), EMBO J. 3, 2613-2619). Characteristic for their aminoacyl acceptor arm is the presence of a so-called pseudoknot which we now also find in a corresponding position at the 3' terminus of TRV RNA2 (PSG strain). The nucleotide sequences of all tobraviral RNAs analysed so far indicate that they all possess a similar 3' terminal structure. A domain resembling the anticodon arm of canonical tRNA is not readily recognizable. TRV RNA2 can be adenylated with CTP, ATP; tRNA nucleotidyl transferase and ATP. It is unable, however, to accept any of the twenty common amino acids when incubated with ATP and aminoacyl-tRNA synthetases from wheat germ or yeast. We conclude that TRV RNA contains a tRNA-like structure, which, in contrast to the tymo- and tobamoviral tRNA-like structures, cannot be aminoacylated. It is unlikely therefore, that aminoacylation of plant viral RNAs with a tRNA-like structure is a prerequisite for viral RNA replication. 相似文献
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《Seminars in Virology》1994,5(1):61-69
Double-stranded RNA viruses have a virion-associated RNA-dependent RNA polymerase activity which is involved in such critical steps of viral assembly as genome packaging and minus strand synthesis. In vitro studies of a bacterial dsRNA virus, ø6, and a yeast virus, L-A, have shed light on capsid formation as well as on the protein/RNA interactions and packaging of the viral genomes. In the ø6 system, an empty dodecahedral polymerase complex (procapsid) composed of four protein species is formed without the help of other viral proteins or RNA. This particle packages positive sense viral RNA genome segments in an ATP dependent reaction. The presence of all rNTPs allows the synthesis of complementary (-) strands within the particle. Self-assembly of an additional protein shell (composed of protein P8) around this particle takes place in the presence of Ca2+ ions. In vivo, these nucleocapsids obtain an envelope while still residing in the cell cytoplasm. L-A, in contrast, is not known to make a prohead structure. The Pol domain of L-A's Gag-Pol fusion protein is necessary for packaging of the (+) strand RNA and probably actually binds to the (+) strand packaging site (a stem-loop with a protruding A) insuring its packaging while the Gag domain primes polymerization of the coat protein. N-Acetylation of Gag by the host MAK3 N-acetyltransferase is necessary for proper assembly, and the ratio of Gag-Pol/Gag, determined by the efficiency of - 1 ribosomal frameshifting, is critical for propagation of the M1 satellite dsRNA. 相似文献
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Nodavirus coat protein imposes dodecahedral RNA structure independent of nucleotide sequence and length
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Tihova M Dryden KA Le TV Harvey SC Johnson JE Yeager M Schneemann A 《Journal of virology》2004,78(6):2897-2905
The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T=3 symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fraction of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex RNA. The similarity in tertiary structure suggests that RNA folding is independent of sequence and length. Computational modeling indicated that RNA duplex formation involves both short-range and long-range interactions. We propose that the capsid protein is able to exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the geometry of the dodecahedral cage. 相似文献
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RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication. 相似文献
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Klug A 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1999,354(1383):531-535
A short account is given of the physical and chemical studies that have led to an understanding of the structure of the tobacco mosaic virus particle and how it is assembled from its constituent coat protein and RNA. The assembly is a much more complex process than might have been expected from the simplicity of the helical design of the particle. The protein forms an obligatory intermediate (a cylindrical disk composed of two layers of protein units), which recognizes a specific RNA hairpin sequence. This extraordinary mechanism simultaneously fulfils the physical requirement for nucleating the growth of the helical particle and the biological requirement for specific recognition of the viral DNA. 相似文献
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Stephen C. Harvey Yingying Zeng Christine E. Heitsch 《Journal of biological physics》2013,39(2):163-172
There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure—often one or more stem-loops—either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these “fingers” of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs. 相似文献
9.
Selection and analysis of mutations in an encephalomyocarditis virus internal ribosome entry site that improve the efficiency of a bicistronic flavivirus construct
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Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as an mRNA for translation of the viral proteins. All of the structural and nonstructural proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (K. K. Orlinger, V. M. Hoenninger, R. M. Kofler, and C. W. Mandl, J. Virol. 80:12197-12208, 2006), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3' noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligo(A) loop of the J-K stem-loop structure, a binding site for the eukaryotic translation initiation factor 4G. These included single-nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligo(A) loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors. 相似文献
10.
Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus. 相似文献
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The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle. 相似文献
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Kickhoefer VA Poderycki MJ Chan EK Rome LH 《The Journal of biological chemistry》2002,277(43):41282-41286
Vaults are highly conserved ubiquitous ribonucleoprotein particles with an undefined function. Three protein species (p240/TEP1, p193/VPARP, and p100/MVP) and a small RNA comprise the 13-MDa vault particle. The expression of the unique 100-kDa major vault protein is sufficient to form the basic vault structure. Previously, we have shown that stable association of the vault RNA with the vault particle is dependent on its interaction with the p240/TEP1 protein. To identify other proteins that interact with the vault RNA, we used a UV-cross-linking assay. We find that a portion of the vault RNA is complexed with the La autoantigen in a separate smaller ribonucleoprotein particle. La interacts with the vault RNA (both in vivo and in vitro) presumably through binding to 3'-uridylates. Moreover, we also demonstrate that the La autoantigen is the 50-kDa protein that we have previously reported as a protein that co-purifies with vaults. 相似文献
13.
S Altamura E Caprini M E Sanchez P Londei 《The Journal of biological chemistry》1991,266(10):6195-6200
Studies of ribosome structure in thermophilic archaebacteria may provide valuable information on (i) the mechanisms involved in the stabilization of nucleic acid-protein complexes at high temperatures and (ii) the degree of evolutionary conservation of the ribosomal components in the primary kingdoms of cell descent. In this work we investigate certain aspects of RNA/protein interaction within the large ribosomal subunits of the extremely thermophilic archaebacterium Sulfolobus solfataricus. The ribosomal proteins involved in the early reactions leading to in vitro particle assembly have been identified; it is shown that they can interact with the RNA in a temperature-independent fashion, forming a thermally stable "core" particle that can subsequently be converted into complete 50 S ribosomes. Among the protein components of the core particle, those capable of independently binding to 23 and 5 S RNA species have also been identified. Finally, we show that the early assembly proteins of Sulfolobus large ribosomal subunits are able to interact cooperatively with 23 S RNAs from other archaebacteria or from eubacteria, thereby suggesting that RNA/protein recognition sites are largely conserved within prokaryotic ribosomes. By contrast, no specific binding of the archaebacterial proteins to eukaryotic RNA could be demonstrated. 相似文献
14.
Specific binding of host cell proteins to the 3''-terminal stem-loop structure of rubella virus negative-strand RNA. 总被引:17,自引:14,他引:3
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At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed. 相似文献
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Cristofari G Ivanyi-Nagy R Gabus C Boulant S Lavergne JP Penin F Darlix JL 《Nucleic acids research》2004,32(8):2623-2631
The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. 相似文献
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Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation. 总被引:15,自引:12,他引:3
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The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly. While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly. In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation. In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation. Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation. These results implicated a linkage between RNA encapsidation and virus particle assembly or release. However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA. These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA. 相似文献