scholarly journals Mutagenic Analysis of the 3′ cis-Acting Elements of the Rubella Virus Genome

1999 ◽  
Vol 73 (4) ◽  
pp. 3386-3403 ◽  
Author(s):  
Min-Hsin Chen ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Infectious Clone ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Host Protein ◽  
Directed Mutagenesis ◽  
Coding Region ◽  
Nucleotide Substitutions ◽  
Mobility Shift ◽  
Stem Loop

ABSTRACT Thermodynamically predicted secondary structure analysis of the 3′-terminal 305 nucleotides (nt) of the rubella virus (RUB) genome, a region conserved in all RUB defective interfering RNAs, revealed four stem-loop (SL) structures; SL1 and SL2 are both located in the E1 coding region, while SL3 and SL4 are within the 59-nt 3′ untranslated region (UTR) preceding the poly(A) tract. SL2 is a structure shown to interact with human calreticulin (CAL), an autoantigen potentially involved in RUB RNA replication and pathogenesis. RNase mapping indicated that SL2 and SL3 are in equilibrium between two conformations, in the second of which the previously proposed CAL binding site in SL2, a U-U bulge, is not formed. Site-directed mutagenesis of the 3′ UTR with a RUB infectious clone, Robo302, revealed that most of the 3′ UTR is required for viral viability except for the 3′-terminal 5 nt and the poly(A) tract, although poly(A) was rapidly regenerated during subsequent replication. Maintenance of the overall SL3 structure, the 11-nt single-stranded sequence between SL3 and SL4, and the sequences forming SL4 were all important for viral viability. Studies on the interaction between host factors and the 3′ UTR showed the formation of three RNA-protein complexes by gel mobility shift assay, and UV-induced cross-linking detected six host protein species, with molecular masses of 120, 80, 66, 55, 48, and 36 kDa, interacting with the 3′ UTR. Site-directed mutagenesis of SL2 by nucleotide substitutions showed that maintenance of SL2 stem rather than the U-U bulge was critical in CAL binding since mutants having the U-U bulge base paired had a similar binding activity for CAL as the native structure whereas mutants having the SL2 stem destabilized had much lower binding activity. However, all of these mutations gave rise to viable viruses when introduced into Robo302, indicating that binding of CAL to SL2 is independent of viral viability.

scholarly journals Involvement of a Nonstructural Protein in Poliovirus Capsid Assembly

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Oluwapelumi O. Adeyemi ◽  
Lee Sherry ◽  
Joseph C. Ward ◽  
Danielle M. Pierce ◽  
Morgan R. Herod ◽  
...  
Keyword(s):  
Early Stage ◽  
Selection Process ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Regulatory Protein ◽  
Lethal Mutation ◽  
Site Directed Mutagenesis ◽  
Coding Region ◽  
Virion Assembly ◽  
Vp1 Protein

ABSTRACTVirus capsid proteins must perform a number of roles. These include self-assembly and maintaining stability under challenging environmental conditions, while retaining the conformational flexibility necessary to uncoat and deliver the viral genome into a host cell. Fulfilling these roles could place conflicting constraints on the innate abilities encoded within the protein sequences. In a previous study, we identified a number of mutations within the capsid-coding sequence of poliovirus (PV) that were established in the population during selection for greater thermostability by sequential treatment at progressively higher temperatures. Two mutations in the VP1 protein acquired at an early stage were maintained throughout this selection procedure. One of these mutations prevented virion assembly when introduced into a wild-type (wt) infectious clone. Here we show, by sequencing beyond the capsid-coding region of the heat-selected virions, that two mutations had arisen within the coding region of the 2A protease. Both mutations were maintained throughout the selection process. Introduction of these mutations into a wt infectious clone by site-directed mutagenesis considerably reduced replication. However, they permitted a low level of assembly of infectious virions containing the otherwise lethal mutation in VP1. The 2Apromutations were further shown to slow the kinetics of viral polyprotein processing, and we suggest that this delay improves the correct folding of the mutant capsid precursor protein to permit virion assembly.IMPORTANCERNA viruses, including poliovirus, evolve rapidly due to the error-prone nature of the polymerase enzymes involved in genome replication. Fixation of advantageous mutations may require the acquisition of complementary mutations which can act in concert to achieve a favorable phenotype. This study highlights a compensatory role of a nonstructural regulatory protein, 2Apro, for an otherwise lethal mutation of the structural VP1 protein to facilitate increased thermal resistance. Studying how viruses respond to selection pressures is important for understanding mechanisms which underpin emergence of resistance and could be applied to the future development of antiviral agents and vaccines.

scholarly journals Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

2000 ◽  
Vol 20 (11) ◽  
pp. 3870-3879 ◽  
Author(s):  
Sheng-Fu Wang ◽  
Stephen Ayer ◽  
William A. Segraves ◽  
Daryl R. Williams ◽  
Alexander S. Raikhel
Keyword(s):  
Dna Binding ◽  
Nuclear Receptors ◽  
Hormone Receptors ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Receptor Complex ◽  
S2 Cells ◽  
Ligand Specificity ◽  
Mobility Shift ◽  
Dna Binding Activity

ABSTRACT The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than theDrosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series ofAedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in AedesEcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

scholarly journals Attenuating Mutations in Coxsackievirus B3 Map to a Conformational Epitope That Comprises the Puff Region of VP2 and the Knob of VP3

2004 ◽  
Vol 78 (24) ◽  
pp. 13987-14002 ◽  
Author(s):  
E. Stadnick ◽  
M. Dan ◽  
A. Sadeghi ◽  
J. K. Chantler
Keyword(s):  
Infectious Clone ◽  
Conformational Epitope ◽  
Coxsackievirus B3 ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Viral Virulence ◽  
Nontranslated Region ◽  
Escape Mutants ◽  
Entire Sequence ◽  
Virulent Phenotype

ABSTRACT Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5′ nontranslated region (5′NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100- to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5′NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5′NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.

scholarly journals Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

2004 ◽  
Vol 186 (3) ◽  
pp. 850-857 ◽  
Author(s):  
Geneviève Renauld-Mongénie ◽  
Laurence Lins ◽  
Tino Krell ◽  
Laure Laffly ◽  
Michèle Mignon ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Binding Protein ◽  
Prediction Method ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Directed Mutagenesis ◽  
Binding Domains ◽  
Homologous Strain ◽  
Protein B

ABSTRACT A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

scholarly journals Structural analysis of natriuretic peptide receptor-C by truncation and site-directed mutagenesis

1997 ◽  
Vol 322 (2) ◽  
pp. 585-590 ◽  
Author(s):  
Makoto ITAKURA ◽  
Hiroyuki SUZUKI ◽  
Shigehisa HIROSE
Keyword(s):  
Ligand Binding ◽  
Natriuretic Peptide ◽  
Gel Filtration ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Natriuretic Peptide Receptor ◽  
Peptide Receptor ◽  
Mammalian Expression ◽  
Ligand Binding Activity

Natriuretic peptide receptor-C (NPR-C) has a unique structure consisting of pre-existing covalent homodimers, but it is not known whether each subunit has ligand-binding activity or whether the dimeric structure is necessary for binding activity. To answer this question, a number of C-terminally truncated mutants were designed, subcloned into the mammalian expression vector pcDNA3 and expressed by transient transfection in COS-1 cells. Truncation at position 461, which eliminates the residue Cys469 that is involved in disulphide-linked dimerization, produced a soluble and monomeric form of NPR-C, as determined by gel filtration on Superose 12. Binding assays of the gel-filtration fractions clearly demonstrated that even monomeric NPR-C contains a high-affinity binding site for natriuretic peptides. Site-directed mutagenesis of the invariant residues (Asp407-Arg408 and Asp411-Phe412) in a region highly conserved among various species established that these invariant residues are essential for ligand-binding activity.

Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis

1987 ◽  
Vol 7 (9) ◽  
pp. 3092-3097
Author(s):  
D J Clanton ◽  
Y Y Lu ◽  
D G Blair ◽  
T Y Shih
Keyword(s):  
Cell Lines ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Soft Agar ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gtp Binding ◽  
Nih 3T3 ◽  
Autokinase Activity

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

scholarly journals Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

2000 ◽  
Vol 74 (10) ◽  
pp. 4590-4600 ◽  
Author(s):  
Ian Goodfellow ◽  
Yasmin Chaudhry ◽  
Andrew Richardson ◽  
Janet Meredith ◽  
Jeffrey W. Almond ◽  
...  
Keyword(s):  
Rna Virus ◽  
Site Directed Mutagenesis ◽  
Evolutionary Significance ◽  
Rna Structures ◽  
Coding Region ◽  
Rna Sequences ◽  
Stem Loop ◽  
Cis Acting ◽  
Function Recovery ◽  
Positive Sense

ABSTRACT The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel polioviruscis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.

scholarly journals La protein binds the predicted loop structures in the 3′ non-coding region of Japanese encephalitis virus genome: role in virus replication

2009 ◽  
Vol 90 (6) ◽  
pp. 1343-1352 ◽  
Author(s):  
Surender Vashist ◽  
Manu Anantpadma ◽  
Himani Sharma ◽  
Sudhanshu Vrati
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Protein Interaction ◽  
Cultured Cells ◽  
Virus Genome ◽  
Encephalitis Virus ◽  
Coding Region ◽  
Mobility Shift ◽  
Stem Loop ◽  
La Protein

Japanese encephalitis virus (JEV) genome is a single-stranded, positive-sense RNA with non-coding regions (NCRs) of 95 and 585 bases at its 5′ and 3′ ends, respectively. These may bind to viral or host proteins important for viral replication. It has been shown previously that three proteins of 32, 35 and 50 kDa bind the 3′ stem–loop (SL) structure of the JEV 3′ NCR, and one of these was identified as 36 kDa Mov34 protein. Using electrophoretic mobility-shift and UV cross-linking assays, as well as a yeast three-hybrid system, it was shown here that La protein binds to the 3′ SL of JEV. The binding was stable under high-salt conditions (300 mM KCl) and the affinity of the RNA–protein interaction was high; the dissociation constant (K d) for binding of La protein to the 3′ SL was 12 nM, indicating that this RNA–protein interaction is physiologically plausible. Only the N-terminal half of La protein containing RNA recognition motifs 1 and 2 interacted with JEV RNA. An RNA toe-printing assay followed by deletion mutagenesis showed that La protein bound to predicted loop structures in the 3′ SL RNA. Furthermore, it was shown that small interfering RNA-mediated downregulation of La protein resulted in repression of JEV replication in cultured cells.

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