scholarly journals Human Rhinovirus Type 14 Gain-of-Function Mutants for oriI Utilization Define Residues of 3C(D) and 3Dpol That Contribute to Assembly and Stability of the Picornavirus VPg Uridylylation Complex

2007 ◽  
Vol 81 (22) ◽  
pp. 12485-12495 ◽  
Author(s):  
Miaoqing Shen ◽  
Qixin Wang ◽  
Yan Yang ◽  
Harsh B. Pathak ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Human Rhinovirus ◽  
Adaptive Mutation ◽  
Sequence Specificity ◽  
Wild Type ◽  
Stem Loop ◽  
Additional Support ◽  
Type 3 ◽  
Enhanced Stability

ABSTRACT VPg linkage to the 5′ ends of picornavirus RNAs requires production of VPg-pUpU. VPg-pUpU is templated by an RNA stem-loop (the cre or oriI) found at different locations in picornavirus genomes. At least one adaptive mutation is required for human rhinovirus type 14 (HRV-14) to use poliovirus type 3 (PV-3) or PV-1 oriI efficiently. One mutation changes Leu-94 of 3C to Pro; the other changes Asp-406 of 3Dpol to Asn. By using an in vitro VPg uridylylation system for HRV-14 that recapitulates biological phenotypes, we show that the 3C adaptive mutation functions at the level of 3C(D) and the 3D adaptive mutation functions at the level of 3Dpol. Pro-94 3C(D) has an expanded specificity and enhanced stability relative to wild-type 3C(D) that leads to production of more processive uridylylation complexes. PV-1/HRV-14 oriI chimeras reveal sequence specificity in 3C(D) recognition of oriI that resides in the upper stem. Asn-406 3Dpol is as active as wild-type 3Dpol in RNA-primed reactions but exhibits greater VPg uridylylation activity due to more efficient recruitment to and retention in the VPg uridylylation complex. Asn-406 3Dpol from PV-1 exhibits identical behavior. These studies suggest a two-step binding mechanism in the assembly of the 3C(D)-oriI complex that leads to unwinding of at least the upper stem of oriI and provide additional support for a direct interaction between the back of the thumb of 3Dpol and 3C that is required for 3Dpol recruitment to and retention in the uridylylation complex.

scholarly journals Sequence Requirements for Viral RNA Replication and VPg Uridylylation Directed by the Internal cis-Acting Replication Element (cre) of Human Rhinovirus Type 14

2002 ◽  
Vol 76 (15) ◽  
pp. 7485-7494 ◽  
Author(s):  
Yan Yang ◽  
Rene Rijnbrand ◽  
Kevin L. McKnight ◽  
Eckard Wimmer ◽  
Aniko Paul ◽  
...  
Keyword(s):  
Rna Structure ◽  
Rna Synthesis ◽  
Mutational Analysis ◽  
Rna Replication ◽  
Human Rhinovirus ◽  
Viral Rna ◽  
Base Substitution ◽  
Stem Loop ◽  
Additional Support

ABSTRACT Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5′ and 3′ noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5′ half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

scholarly journals Mutations in PneumococcalcpsEGenerated viaIn VitroSerial Passaging Reveal a Potential Mechanism of Reduced Encapsulation Utilized by a Conjunctival Isolate

2015 ◽  
Vol 197 (10) ◽  
pp. 1781-1791 ◽  
Author(s):  
Mara G. Shainheit ◽  
Michael D. Valentino ◽  
Michael S. Gilmore ◽  
Andrew Camilli
Keyword(s):  
Streptococcus Pneumoniae ◽  
Invasive Disease ◽  
Adaptive Mutation ◽  
Specific Gene ◽  
High Dose ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Sepsis Model

ABSTRACTThe polysaccharide capsule ofStreptococcus pneumoniaeis required for nasopharyngeal colonization and for invasive disease in the lungs, blood, and meninges. In contrast, the vast majority of conjunctival isolates are acapsular. The first serotype-specific gene in the capsule operon,cpsE, encodes the initiating glycosyltransferase and is one of the few serotype-specific genes that can tolerate null mutations. This report characterizes a spontaneously arising TIGR4 mutant exhibiting a reduced capsule, caused by a 6-nucleotide duplication incpsEwhich results in duplication of Ala and Ile at positions 45 and 46. This strain (AI45dup) possessed more exposed phosphorylcholine and was hypersusceptible to C3 complement deposition compared to the wild type. Accordingly, the mutant was significantly better at forming abiotic biofilms and binding epithelial cellsin vitrobut was avirulent in a sepsis model.In vitroserial passaging of the wild-type strain failed to reproduce the AI45dup mutation but instead led to a variety of mutants with reduced capsule harboring single nucleotide polymorphisms (SNPs) incpsE. A single passage in the sepsis model after high-dose inoculation readily yielded revertants of AI45dup with restored wild-type capsule level, but the majority of SNP alleles ofcpsEcould not revert, suppress, or bypass. Analysis ofcpsEin conjunctival isolates revealed a strain with a single missense mutation at amino acid position 377, which was responsible for reduced encapsulation. This study supports the hypothesis that spontaneous, nonreverting mutations incpsEserve as a form of adaptive mutation by providing a selective advantage toS. pneumoniaein niches where expression of capsule is detrimental.IMPORTANCEWhile the capsule ofStreptococcus pneumoniaeis required for colonization and invasive disease, most conjunctival isolates are acapsular by virtue of deletion of the entire capsular operon. We show that spontaneous acapsular mutants isolatedin vitroharbor mostly nonrevertible single nucleotide polymorphism (SNP) null mutations incpsE, encoding the initiating glycosyltransferase. From a small collection of acapsular conjunctival isolates, we identified one strain with a complete capsular operon but containing a SNP incpsEthat we show is responsible for the acapsular phenotype. We propose that acapsular conjunctival isolates may arise initially from such nonreverting SNP null mutations incpsE, which can be followed later by deletion of portions or all of thecpsoperon.

scholarly journals Genetic and Biochemical Studies of Polioviruscis-Acting Replication Element cre in Relation to VPg Uridylylation

2000 ◽  
Vol 74 (22) ◽  
pp. 10371-10380 ◽  
Author(s):  
Elizabeth Rieder ◽  
Aniko V. Paul ◽  
Dong Wook Kim ◽  
Jacques H. van Boom ◽  
Eckard Wimmer
Keyword(s):  
Genome Replication ◽  
Coding Region ◽  
Stem Loop ◽  
Genetic Changes ◽  
Poliovirus Type ◽  
Type 3 ◽  
Type Sequence

ABSTRACT In addition to highly conserved stem-loop structures located in the 5′- and 3′-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941–1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to thecre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3Dpol in a reaction that is greatly stimulated by 3CDpro (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359–10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conservedAAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild-type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.

scholarly journals 3' processing of human pre-U2 small nuclear RNA: a base-pairing interaction between the 3' extension of the precursor and an internal region.

1997 ◽  
Vol 17 (12) ◽  
pp. 7178-7185 ◽  
Author(s):  
Q Huang ◽  
M R Jacobson ◽  
T Pederson
Keyword(s):  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Base Pairing ◽  
Pairing Interaction ◽  
Stem Loop ◽  
Internal Region ◽  
Base Pairing Interaction

The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3' ends. The 3' processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3' processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3' ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3' end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3' processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3' extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3' extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3' processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3' processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3' extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3' processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3' processing of human U2 RNA.

scholarly journals Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNATrpPrimer Annealing

1999 ◽  
Vol 73 (8) ◽  
pp. 6307-6318 ◽  
Author(s):  
Shannon Morris ◽  
Jonathan Leis
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Primer Binding Site ◽  
Wild Type ◽  
Stem Loop ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Primer Annealing ◽  
Nuclease Mapping

ABSTRACT Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

scholarly journals Ebola Virus VP30 Is an RNA Binding Protein

2007 ◽  
Vol 81 (17) ◽  
pp. 8967-8976 ◽  
Author(s):  
Sinu P. John ◽  
Tan Wang ◽  
Scott Steffen ◽  
Sonia Longhi ◽  
Connie S. Schmaljohn ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Ebola Virus ◽  
Rna Binding ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Binding Activity ◽  
Stem Loop ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

ABSTRACT The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

scholarly journals Phenazine-1-Carboxylic Acid Production by Pseudomonas fluorescens LBUM636 Alters Phytophthora infestans Growth and Late Blight Development

2017 ◽  
Vol 107 (3) ◽  
pp. 273-279 ◽  
Author(s):  
Christopher K. Morrison ◽  
Tanya Arseneault ◽  
Amy Novinscak ◽  
Martin Filion
Keyword(s):  
Carboxylic Acid ◽  
Late Blight ◽  
Plant Defense ◽  
Phytophthora Infestans ◽  
Pseudomonas Fluorescens ◽  
Quantitative Polymerase Chain Reaction ◽  
Direct Interaction ◽  
Wild Type ◽  
In Planta

Phytophthora infestans causes late blight of potato, one of the most devastating diseases affecting potato production. Alternative approaches for controlling late blight are being increasingly sought due to increasing environmental concerns over the use of chemical pesticides and the increasing resistance of P. infestans to fungicides. Our research group has isolated a new strain of Pseudomonas fluorescens (LBUM636) of biocontrol interest producing the antibiotic phenazine-1-carboxylic acid (PCA). Wild-type LBUM636 was shown to significantly inhibit the growth of Phytophthora infestans in in vitro confrontational assays whereas its isogenic mutant (phzC−; not producing PCA) only slightly altered the pathogen’s growth. Wild-type LBUM636 but not the phzC− mutant also completely repressed disease symptom development on tubers. A pot experiment revealed that wild-type LBUM636 can significantly reduce P. infestans populations in the rhizosphere and in the roots of potato plants, as well as reduce in planta disease symptoms due to PCA production. The expression of eight common plant defense-related genes (ChtA, PR-1b, PR-2, PR-5, LOX, PIN2, PAL-2, and ERF3) was quantified in tubers, roots, and leaves by reverse-transcription quantitative polymerase chain reaction and revealed that the biocontrol observed was not associated with the induction of a plant defense response by LBUM636. Instead, a direct interaction between P. infestans and LBUM636 is required and PCA production appears to be a key factor for LBUM636’s biocontrol ability.

scholarly journals Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.

1995 ◽  
Vol 15 (11) ◽  
pp. 5966-5974 ◽  
Author(s):  
M Oelgeschläger ◽  
J Krieg ◽  
J M Lüscher-Firzlaff ◽  
B Lüscher
Keyword(s):  
Dna Binding ◽  
Phosphorylation Site ◽  
Direct Interaction ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Wild Type ◽  
Type C ◽  
A Site

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.

scholarly journals A Single Tyrosine in the Severe Acute Respiratory Syndrome Coronavirus Membrane Protein Cytoplasmic Tail Is Important for Efficient Interaction with Spike Protein

2009 ◽  
Vol 84 (4) ◽  
pp. 1891-1901 ◽  
Author(s):  
Corrin E. McBride ◽  
Carolyn E. Machamer
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Direct Interaction ◽  
Cytoplasmic Tail ◽  
Wild Type ◽  
Golgi Region ◽  
Sequence Requirements ◽  
Assembly Site

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 3 major envelope proteins: spike (S), membrane (M), and envelope (E). Previous work identified a dibasic endoplasmic reticulum retrieval signal in the cytoplasmic tail of SARS-CoV S that promotes efficient interaction with SARS-CoV M. The dibasic signal was shown to be important for concentrating S near the virus assembly site rather than for direct interaction with M. Here, we investigated the sequence requirements of the SARS-CoV M protein that are necessary for interaction with SARS-CoV S. The SARS-CoV M tail was shown to be necessary for S localization in the Golgi region when the proteins were exogenously coexpressed in cells. This was specific, since SARS-CoV M did not retain an unrelated glycoprotein in the Golgi. Importantly, we found that an essential tyrosine residue in the SARS-CoV M cytoplasmic tail, Y195, was important for S-M interaction. When Y195 was mutated to alanine, MY195A no longer retained S intracellularly at the Golgi. Unlike wild-type M, MY195A did not reduce the amount of SARS-CoV S carbohydrate processing or surface levels when the two proteins were coexpressed. Mutating Y195 also disrupted SARS-CoV S-M interaction in vitro. These results suggest that Y195 is necessary for efficient SARS-CoV S-M interaction and, thus, has a significant involvement in assembly of infectious virus.

scholarly journals The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5′ structure element

2019 ◽  
Vol 116 (32) ◽  
pp. 15901-15906 ◽  
Author(s):  
Cédric Romilly ◽  
Sebastian Deindl ◽  
E. Gerhart H. Wagner
Keyword(s):  
Ribosomal Protein ◽  
Direct Interaction ◽  
In Vitro Translation ◽  
Rna Structures ◽  
Long Distance ◽  
Stem Loop ◽  
Ribosomal Protein S1 ◽  
Ribosome Binding Sites ◽  
Single Stranded Region

In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking–immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5′-end stem-loop structure. Removal of the latter by 5′-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

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