scholarly journals RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 968 ◽  
Author(s):  
Hyejeong Kim ◽  
Victor D. Ellis ◽  
Andrew Woodman ◽  
Yan Zhao ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Double Mutant ◽  
Biochemical Properties ◽  
Model System ◽  
Viral Population ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Rna Dependent Rna Polymerase ◽  
Or Efficiency ◽  
Coding Sequence

Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.

scholarly journals RNA-dependent RNA polymerase speed and fidelity are not the only determinants of the mechanism or efficiency of recombination

2019 ◽  
Author(s):  
Hyejeong Kim ◽  
Victor D. Ellis ◽  
Andrew Woodman ◽  
Yan Zhao ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Biochemical Property ◽  
Biochemical Parameter ◽  
Biochemical Properties ◽  
Viral Population ◽  
Universal Method ◽  
Wild Type ◽  
Emerging Viruses ◽  
Rna Dependent Rna Polymerase ◽  
Or Efficiency

AbstractUsing the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.SignificanceThe availability of a “universal” method to create attenuated viruses for use as vaccine strains would permit a rapid response to outbreaks of newly emerging viruses. Targeting RdRp fidelity has emerged as such a universal approach. However, because polymerase fidelity and speed are inextricably linked, the effort to attribute the attenuated phenotype to a single biochemical property of the RdRp may be futile. Here, we show that this circumstance is even more complex. We provide evidence for the existence of a biochemical parameter that combines with fidelity and speed to govern the mechanism and/or efficiency of recombination. We conclude that the field will be served best by continued emphasis on discovery of manipulatable functions of the RdRp instead of debating the importance of individual properties.

scholarly journals Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Virus Like Particles ◽  
Rna Polymerase Activity ◽  
Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

scholarly journals New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation

1989 ◽  
Vol 260 (2) ◽  
pp. 491-497 ◽  
Author(s):  
L Hederstedt ◽  
L O Hedén
Keyword(s):  
Bacillus Subtilis ◽  
Succinate Dehydrogenase ◽  
Active Site ◽  
Amino Acid Position ◽  
Biochemical Properties ◽  
Cysteine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Succinate Oxidation ◽  
E Coli

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

scholarly journals Ctr9, Rtf1, and Leo1 Are Components of the Paf1/RNA Polymerase II Complex

2002 ◽  
Vol 22 (7) ◽  
pp. 1971-1980 ◽  
Author(s):  
Cherie L. Mueller ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Double Mutant ◽  
Affinity Purification ◽  
Tata Binding Protein ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Pol Ii ◽  
Paf1 Complex ◽  
Transcriptional Start Sites

ABSTRACT The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Δ phenotypes. In particular, an rtf1Δ paf1Δ double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Δ single mutant. In addition, expression of the G1 cyclin CLN1, reduced nearly threefold in paf1Δ, is restored to wild-type levels in the rtf1Δ paf1Δ double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.

scholarly journals Neutralising antibodies in Spike mediated SARS-CoV-2 adaptation

2020 ◽  
Author(s):  
SA Kemp ◽  
DA Collier ◽  
R Datir ◽  
IATM Ferreira ◽  
S Gayed ◽  
...  
Keyword(s):  
Double Mutant ◽  
Spike Protein ◽  
Viral Population ◽  
Wild Type ◽  
Theoretical Possibility ◽  
Neutralising Antibodies ◽  
Plasma Therapy ◽  
Virus Escape ◽  
Long Read

AbstractSARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against ΔH69/ΔV70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

Seizure Suppression by shakB2, a Gap Junction Mutation in Drosophila

2006 ◽  
Vol 95 (2) ◽  
pp. 627-635 ◽  
Author(s):  
Juan Song ◽  
Mark A. Tanouye
Keyword(s):  
Gap Junction ◽  
Double Mutant ◽  
Model System ◽  
Genetic Interactions ◽  
High Threshold ◽  
Seizure Susceptibility ◽  
Wild Type ◽  
Giant Fiber ◽  
Seizure Disorders ◽  
Junction Proteins

Gap junction proteins mediate electrical synaptic transmission. In Drosophila, flies carrying null mutations in the shakB locus, such as shakB2, have behavioral and electrophysiological defects in the giant fiber (GF) system neurocircuit consistent with a loss of transmission at electrical synapses. The shakB2 mutation also affects seizure susceptibility. Mutant flies are especially seizure-resistant and have a high threshold to evoked seizures. In addition, in some double mutant combinations with “epilepsy” mutations, shakB2 appears to act as a seizure-suppressor mutation: shakB2 restores seizure susceptibility to the wild-type range in the double mutant. In double mutant combinations, shakB2 completely suppresses seizures caused by slamdance ( sda) , knockdown ( kdn), and jitterbug ( jbug) mutations. Seizures caused by easily shocked ( eas) and technical knockout ( tko) mutations are partially suppressed by shakB2. Seizures caused by bang-sensitive ( bas2) and bang- senseless ( bss1, bss2 alleles) mutations are not suppressed by shakB2. These results show the use of Drosophila as a model system for studying the kinds of genetic interactions responsible for seizure susceptibility, bringing us closer to unraveling the complexity of seizure disorders in humans.

scholarly journals Development of a Challenge-Protective Vaccine Concept by Modification of the Viral RNA-Dependent RNA Polymerase of Canine Distemper Virus

2007 ◽  
Vol 81 (24) ◽  
pp. 13649-13658 ◽  
Author(s):  
D. Silin ◽  
O. Lyubomska ◽  
M. Ludlow ◽  
W. P. Duprex ◽  
B. K. Rima
Keyword(s):  
Rna Polymerase ◽  
Neutralizing Antibodies ◽  
Canine Distemper Virus ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Hinge Region ◽  
Canine Distemper ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
L Protein

ABSTRACT We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a “quick response” in vaccine development against well-known and “new” viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.

Enhancement of Acid Stability of Alpha Amylase from Bacillus licheniformis by Error-Prone PCR

2013 ◽  
Vol 774-776 ◽  
pp. 664-669
Author(s):  
Yan Jing Xu ◽  
Yi Han Liu ◽  
Shuai Fan ◽  
Fu Ping Lu
Keyword(s):  
Bacillus Licheniformis ◽  
Double Mutant ◽  
Kinetic Studies ◽  
Site Directed Mutagenesis ◽  
Alpha Amylase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Chain Reaction ◽  
Acid Stability ◽  
Polymerase Chain

Acid stability of Bacillus licheniformis alpha amylase (BLA) was improved by error-prone polymerase chain reaction. The mutated BLA gene was expressed in Escherichia coli. An acid stability double mutant (K344R/H405R in BLA) was isolated. Two single mutants K344R and H405R were obtained by the way of site-directed mutagenesis. The enzymes (BLA) of the three mutants were isolated and characterized. Kinetic studies showed that the kcat/Km values of the mutants K344R, H405R, and K344R/H405R under pH 4.5 were about 8-, 11.5-, and 17.7-times higher than that of the wild type enzyme. As revealed by the structure models of the wild-type and mutant enzymes, the amino acids substituted of R344 and R405 in the BLA contribute to its acid stability.

scholarly journals Mechanism of Activation of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

2007 ◽  
Vol 51 (2) ◽  
pp. 503-509 ◽  
Author(s):  
Eisuke Murakami ◽  
Haiying Bao ◽  
Mangala Ramesh ◽  
Tamara R. McBrayer ◽  
Tony Whitaker ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Specific Inhibitor ◽  
Nucleoside Diphosphate Kinase ◽  
Hcv Rna ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Hcv Ns5b

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 μM and a k cat of 0.007 s−1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (Ki = 1.5 μM) and 2′-C-methylcytidine triphosphate (Ki = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.

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