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1.
In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required. 相似文献
2.
As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies. 相似文献
3.
A genetic locus in mutant poliovirus genomes involved in overproduction of RNA polymerase and 3C proteinase 总被引:2,自引:0,他引:2
A mutagenic oligonucleotide cassette was used to introduce single and tandem amino acid substitutions into the proteinase 3C coding region of an infectious poliovirus type 1 cDNA. The sites targeted for mutagenesis, residues 60, 61, and 66, are located within a putative helical loop structure which may be involved in substrate recognition by the enzyme. Fourteen viable 3C proteinase mutants were isolated. A Lys----Arg substitution at position 60 resulted in cold sensitivity for growth at 33 degrees. Replacement of Lys 60 with Ile, either singly or in combination with substitutions at position 61, resulted in viruses that produced three- to fivefold more 3D RNA polymerase than wild-type poliovirus. 3C-mediated processing of the remaining sites within the polyprotein was not noticeably affected. The overproduction of 3D is a consequence of more efficient processing of the carboxy-terminal Gln-Gly amino acid pair of 3C. Together with a previous report in which substitution of Val 54 with an Ala residue results in a poliovirus that produces decreased levels of 3D, these observations provide evidence that the putative loop region (residues 51-66) may be a functional domain involved in recognition of the carboxy-terminal Gln-Gly cleavage site of 3C. 相似文献
4.
Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5′ noncoding region (5′ NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5′ NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody. 相似文献
5.
Determinants of temperature sensitivity and/or attenuation in Sabin type 1 poliovirus reside in the 5' NTR and coding sequences of the capsid proteins and viral RNA polymerase, 3D(pol). Previous studies have implicated at least two mutations in 3D(pol) of Sabin 1 vaccine strain [PV1(S)], including a Y73H change, as contributing to these phenotypes. We have used an in vitro assay to test the first step in RNA synthesis, the uridylylation of the terminal protein VPg with 3D(pol) isolated from PV1(S). Wt and two mutant 3D(pol) proteins (Y73H, D53N/Y73H) were expressed in Escherichia coli and were purified, and their activities were measured in the synthesis of VPgpU(pU) and of VPg-linked poly(U) at 30 and 39.5 degrees C. Our results show that at 39.5 degrees C the Y73H mutation leads to a defect in the synthesis of VPgpUp(U) and of VPg-poly(U) but not in the elongation of a (dT)(15) primer. The double mutant protein had the same activities as Y73H 3D(pol). Using the yeast two-hybrid assay, we detected a reduced interaction between 3D(pol) molecules carrying either the single or double mutations. Tyrosine-73 maps to the finger domain in the three-dimensional structure of 3D(pol). A model will be presented in which a change of Y73 to H73 may interfere with an interaction between two polymerase molecules that, in turn, may interfere with VPg uridylylation. Alternative explanations, however, cannot be excluded at the present time. 相似文献
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A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic. 总被引:2,自引:2,他引:2
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A D Murdin H Su D S Manning M H Klein M J Parnell H D Caldwell 《Infection and immunity》1993,61(10):4406-4414
Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines. 相似文献
9.
Defined recombinants of poliovirus and coxsackievirus: sequence-specific deletions and functional substitutions in the 5'-noncoding regions of viral RNAs 总被引:15,自引:0,他引:15
We describe the isolation of a variant of a polio--coxsackie recombinant virus (PCV110) containing a genomic RNA with a chimeric 5'-noncoding region. The variant virus [designated PCV110(1)] has growth and biosynthetic properties that are quite different from the original, temperature-sensitive isolate of the recombinant virus [designated PCV110(4)]. Nucleotide sequencing of the 5'-noncoding region of RNA from PCV110(1) revealed a 4-base deletion within the substituted coxsackievirus region of the chimeric genome that may contribute to the loss of temperature sensitivity of this variant recombinant virus. In addition, we have generated new recombinant viruses that contain (1) coxsackievirus sequences within the N66-N627 region of the poliovirus genome and (2) coxsackievirus sequences substituted from N1-N627 in the poliovirus genome. These recombinant viruses are not temperature sensitive for growth at 37 degrees and have biosynthetic properties similar to those of wild-type poliovirus. Our results provide evidence that replicase recognition signals encoded in the 5' noncoding regions of enterovirus genomic RNAs are not strictly sequence specific. 相似文献
10.
Tohru Daikoku Mineyuki Mizuguchi Takayuki Obita Takeshi Yokoyama Yoshihiro Yoshida Masaya Takemoto Kimiyasu Shiraki 《Journal of microbiology, immunology, and infection》2018,51(5):581-586
Background
T-705 (favipiravir) is a potent inhibitor of RNA-dependent RNA polymerases of influenza viruses and no favipiravir-resistant virus has been isolated. Poliovirus RNA polymerase has been well characterized and isolation of resistant virus was examined in poliovirus.Methods
Susceptibility variants of poliovirus I (Sabin strain) were isolated during passages in the presence of favipiravir and characterized for their susceptibility and the sequence of RNA polymerase.Results
Five variants with 0.47–1.88 times the 50% inhibitory concentration for plaque formation of the parent poliovirus had amino acid variations in the 3D gene of the RNA polymerase. The distribution of amino acid variations was not related to ribavirin resistance, and two amino acid variation sites were found near the finger domain.Conclusion
Favipiravir as a chain terminator would not be incorporated and replicate to cause lethal mutagenesis as a mutagen like ribavirin, and resistant mutants were not isolated. A high replication level would generate mutations leading to favipiravir resistance as ribavirin resistance was generated, but generated mutations would be lethal to the RNA polymerase function. 相似文献11.
T Y Nakagawa H Von Grafenstein J E Sears J Williams C A Janeway R A Flavell 《European journal of immunology》1991,21(11):2851-2855
CD4+ T cells recognize processed exogenous antigen in the form of peptides bound to syngeneic major histocompatibility complex class II molecules on antigen-presenting cells. We have developed a novel and convenient method to synthesize and map CD4+ T cell epitopes of cloned antigens using polymerase chain reaction (PCR)-directed construction of genes expressing recombinant protein fragments. Unique restriction sites incorporated into the PCR primers were employed for the unidirectional cloning of gene fragments into a bacterial expression vector that can be induced to high-level expression. The bacterial lysate could be used directly in T cell proliferation assays. Overlapping recombinant fragments spanning the entire protein were generated and tested. The length of the sequence containing the epitope was further reduced by utilizing PCR to generate 3' truncations. Finally, a small number of overlapping peptides spanning a sequence of 39 amino acids were synthesized to identify a thirteen-amino acid peptide epitope within chicken transferrin that stimulates the T helper cell clone D10.G4.1. PCR-directed construction of fragments of antigen allows for optimal design of strategies for the mapping and analysis of CD4+ T cell epitopes. 相似文献
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Previous results from our laboratory have identified a small (60 nt) RNA from the yeast S. cerevisiae that specifically inhibits internal ribosome entry site (IRES)-mediated translation programmed by poliovirus (PV) and hepatitis C virus (HCV) 5'-untranslated region (5'UTR). The yeast inhibitor RNA (called IRNA) was found to efficiently compete with viral 5'UTR for binding of several cellular polypeptides that presumably play important roles in IRES-mediated translation. One such IRNA (and 5'UTR)-binding protein has previously been identified as the La autoantigen. In this report, we have identified a 110-kDa IRNA-binding protein (which also interacts with viral 5'UTR) as nucleolin, a nucleolar RNA binding protein that was previously shown to translocate into the cytoplasm following infection of cells with poliovirus. We demonstrate that nucleolin (called C23) stimulates viral IRES-mediated translation both in vitro and in vivo. We also show that nucleolin mutants containing the carboxy-terminal RNA binding domains but lacking the amino terminal domain inhibit IRES-mediated translation in vitro. The translation inhibitory activity of these mutants correlates with their ability to bind the 5'UTR sequence. These results suggest a role of nucleolin/C23 in viral IRES-mediated translation. 相似文献
13.
Southern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1--57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an alpha-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed. 相似文献
14.
The cDNA coding sequence of the RNA-dependent RNA polymerase (3Dpol) of Mengovirus was cloned and expressed in a bacterial system. Eleven monoclonal antibodies were raised against the recombinant Mengo 3Dpol (rM3D). All of them recognized the recombinant and the viral-induced form of the protein. The panel of monoclonals belonged to the IgG1 and IgG2a isotypes and were mapped to four different epitopes in the 3D molecule by competition assays. All monoclonals recognized Mengo 3Dpol in western blots and cross-reacted with the homologous polymerases of seven other cardioviruses but failed to react with 3Dpol from poliovirus type 1 and 3 or rhinovirus type 14 and 16. 相似文献
15.
Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system 总被引:2,自引:0,他引:2
The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible. 相似文献
16.
In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. 相似文献
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Kittel C Sereinig S Ferko B Stasakova J Romanova J Wolkerstorfer A Katinger H Egorov A 《Virology》2004,324(1):67-73
In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice. 相似文献