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 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 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 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.

scholarly journals Venezuelan Equine Encephalitis Virus V3526 Vaccine RNA-Dependent RNA Polymerase Mutants Increase Vaccine Safety Through Restricted Tissue Tropism in a Mouse Model

Zoonoses ◽  
2022 ◽  
Vol 2 (1) ◽  
Author(s):  
Clint A. Haines ◽  
Rafael K. Campos ◽  
Sasha R. Azar ◽  
K. Lane Warmbrod ◽  
Tiffany F. Kautz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Vaccine Efficacy ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Effective Vaccine ◽  
Tissue Tropism ◽  
Venezuelan Equine Encephalitis ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Equine Encephalitis Virus

Background: Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas, for which no vaccines or antiviral agents have been approved. TC-83 and V3526 are the best-characterized vaccine candidates for VEEV. Both are live-attenuated vaccines and have been associated with safety concerns, although fewer concerns exist for V3526. A previous attempt to improve the TC-83 vaccine focused on further attenuating the vaccine by adding mutations that alter the error-incorporation rate of the RNA-dependent RNA polymerase (RdRp). Methods: The research herein examined the effects of these RdRp mutations in V3526 by cloning the 3X and 4X strains, assessing vaccine efficacy against challenge in adult female CD-1 mice, examining neutralizing-antibody titers, investigating vaccine tissue tropism, and testing the stability of the mutant strains. Results: The V3526 RdRp mutants exhibited less tissue tropism in the spleen and kidney than the wild-type V3526, while maintaining vaccine efficacy. Illumina sequencing indicated that the RdRp mutations reverted to wild-type V3526 after five passages in murine pup brains. Conclusions: The observed genotypic reversion is likely to be of limited concern, because wild-type V3526 remains an effective vaccine capable of providing protection. Our results indicate that the V3526 RdRp mutants may be a safer vaccine design than the original V3526.

scholarly journals The Delta Subunit of RNA Polymerase, RpoE, Is a Global Modulator of Streptococcus mutans Environmental Adaptation

2010 ◽  
Vol 192 (19) ◽  
pp. 5081-5092 ◽  
Author(s):  
Xiaoli Xue ◽  
Jürgen Tomasch ◽  
Helena Sztajer ◽  
Irene Wagner-Döbler
Keyword(s):  
Hydrogen Peroxide ◽  
Rna Polymerase ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Transcriptome Profiling ◽  
Biochemical Properties ◽  
Environmental Adaptation ◽  
Wild Type ◽  
Delta Subunit

ABSTRACT The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild-type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ΔrpoE mutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans which is required for optimal growth and environmental adaptation.

Selection of a Thiazole Urea-Resistant Variant of Bovine Viral Diarrhoea Virus That Maps to the RNA-Dependent RNA Polymerase

2002 ◽  
Vol 13 (5) ◽  
pp. 315-323 ◽  
Author(s):  
Robert W King ◽  
Helen T Scarnati ◽  
E Scott Priestley ◽  
Indawati De Lucca ◽  
Anu Bansal ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bovine Viral Diarrhoea Virus ◽  
Fold Increase ◽  
Bovine Viral Diarrhoea ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Resistant Variant ◽  
Diarrhoea Virus ◽  
Template Selection ◽  
Selection Of

By passing wild type bovine viral diarrhoea virus (BVDV) in increasing concentrations of DPC-A69280–29, a thiazole urea class compound that inhibits BVDV replication, we were able to select several variants of BVDV that exhibited decreased susceptibility to this compound. When the non-structural genes of these variants were sequenced and compared with wild type, only one change was common to all the variants that also exhibited resistance to DPC-A69280–29 (>10-fold increase in IC50). This change was a T-to-A transversion at position 11198 of the BVDV genome, which would cause a predicted substitution of isoleucine for phenylalanine at amino acid 78 of the RNA-dependent RNA polymerase (RdRp). This substitution would occur in a region of the BVDV RdRp which has been proposed to be important for the formation of the RdRp homodimer that is essential for the activity of the enzyme. However, since DPC-69280-29 inhibits BVDV replication by interfering with the initiation of viral RNA synthesis, we discuss the possibility that this region of the BVDV RdRp also may play a role in the initiation process. Furthermore, since this region is located fairly close to the template RNA, we also propose that the role it plays may involve either template selection, stabilization or processivity.

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 Catalytic Core of Alphavirus Nonstructural Protein nsP4 Possesses Terminal Adenylyltransferase Activity

2006 ◽  
Vol 80 (20) ◽  
pp. 9962-9969 ◽  
Author(s):  
Shailly Tomar ◽  
Richard W. Hardy ◽  
Janet L. Smith ◽  
Richard J. Kuhn
Keyword(s):  
Rna Polymerase ◽  
Sindbis Virus ◽  
Catalytic Domain ◽  
Function Analysis ◽  
Nonstructural Protein ◽  
Divalent Cations ◽  
Viral Rna ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Catalytic Core

ABSTRACT The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in Escherichia coli. The core catalytic domain of nsP4 (Δ97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that Δ97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3′ end of an acceptor RNA in the presence of divalent cations. Furthermore, Δ97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. Δ97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to ∼45% of that of the wild type in the presence of Mg2+. Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn2+. Identification of Δ97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.

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