scholarly journals The cost of replication fidelity in human immunodeficiency virus type 1

2006 ◽  
Vol 274 (1607) ◽  
pp. 225-230 ◽  
Author(s):  
Victoria Furió ◽  
Andrés Moya ◽  
Rafael Sanjuán
Keyword(s):  
Rna Viruses ◽  
Mutation Rates ◽  
Published Data ◽  
Replication Fidelity ◽  
Biochemical Basis ◽  
Trade Off ◽  
The Cost ◽  
Micro Organisms ◽  
Hiv 1

Mutation rates should be governed by at least three evolutionary factors: the need for beneficial mutations, the benefit of minimizing the mutational load and the cost of replication fidelity. RNA viruses show high mutation rates compared with DNA micro-organisms, and recent findings suggest that the cost of fidelity might play a role in the evolution of increased mutation rates. Here, by analysing previously published data from HIV-1 reverse transcriptase in vitro assays, we show a trade-off between enzymatic accuracy and the maximum rate of polymerization, thus providing a biochemical basis for the fitness cost of fidelity in HIV-1. This trade-off seems to be related to inefficient extension of mispairs, which increases fidelity at the expense of the polymerization rate. Since in RNA viruses fast replication is critical for survival, this could impose a high cost of fidelity and favour the evolution of high mutation rates.

scholarly journals Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

2014 ◽  
Vol 89 (1) ◽  
pp. 275-286 ◽  
Author(s):  
Grace Campagnola ◽  
Seth McDonald ◽  
Stéphanie Beaucourt ◽  
Marco Vignuzzi ◽  
Olve B. Peersen
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Mutation Rates ◽  
Rapid Adaptation ◽  
Replication Fidelity ◽  
Virus Growth ◽  
Polymerase Structure ◽  
Positive Strand Rna

ABSTRACTViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates andin vivofidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects onin vitronucleotide discrimination, and these effects are strongly correlated with elongation rates andin vivomutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects onin vitronucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growthin vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

scholarly journals Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9864-9878 ◽  
Author(s):  
Michael E. Abram ◽  
Andrea L. Ferris ◽  
Wei Shao ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Viral Replication ◽  
Mutation Rate ◽  
Hot Spots ◽  
High Rate ◽  
Published Data ◽  
Single Cycle ◽  
Efficient System ◽  
Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

scholarly journals Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

2017 ◽  
Vol 115 (2) ◽  
pp. E162-E171 ◽  
Author(s):  
François Ferron ◽  
Lorenzo Subissi ◽  
Ana Theresa Silveira De Morais ◽  
Nhung Thi Tuyet Le ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Mutation Rates ◽  
Bifunctional Enzyme ◽  
Antiviral Compound ◽  
Mismatch Correction ◽  
Limited Efficacy

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

scholarly journals Spontaneous Mutations in HIV-1 Gag, Protease, RT p66 in the First Replication Cycle and How They Appear: Insights from an In Vitro Assay on Mutation Rates and Types

2020 ◽  
Vol 22 (1) ◽  
pp. 370
Author(s):  
Joshua Yi Yeo ◽  
Darius Wen-Shuo Koh ◽  
Ping Yap ◽  
Ghin-Ray Goh ◽  
Samuel Ken-En Gan
Keyword(s):  
Codon Usage ◽  
Selection Pressure ◽  
In Vitro Selection ◽  
Mutation Rates ◽  
Free Energies ◽  
Vitro Assay ◽  
Free System ◽  
Functional Sites ◽  
Hiv 1

While drug resistant mutations in HIV-1 are largely credited to its error prone HIV-1 RT, the time point in the infection cycle that these mutations can arise and if they appear spontaneously without selection pressures both remained enigmatic. Many HIV-1 RT mutational in vitro studies utilized reporter genes (LacZ) as a template to investigate these questions, thereby not accounting for the possible contribution of viral codon usage. To address this gap, we investigated HIV-1 RT mutation rates and biases on its own Gag, protease, and RT p66 genes in an in vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease, and RT p66 at 4.71 × 10−5, 6.03 × 10−5, and 7.09 × 10−5 mutations/bp, respectively. Gag and p66 genes showed a large number of ‘A to G’ mutations. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 × 10−4 mutations/bp) and that ‘A to G’ mutations occurred in regions reminiscent of ADAR neighbor sequence preferences. Mutational free energies of the ‘A to G’ mutations revealed an avoidance of destabilizing effects, with the natural p66 gene codon usage providing barriers to disruptive amino acid changes. Our study demonstrates the importance of studying mutation emergence in HIV genes in a RT-PCR in vitro selection pressure free system to understand how fast drug resistance can emerge, providing transferable applications to how new viral diseases and drug resistances can emerge.

scholarly journals Low recombination activity of R region located at both ends of the HIV-1 genome.

2012 ◽  
Vol 59 (4) ◽  
Author(s):  
Anna Urbanowicz ◽  
Anna Kurzyńska-Kokorniak ◽  
Anna Jankowska ◽  
Magdalena Alejska ◽  
Marek Figlerowicz
Keyword(s):  
Rna Viruses ◽  
Brome Mosaic Virus ◽  
Template Switching ◽  
Strand Transfer ◽  
Recombination Activity ◽  
Rna Sequences ◽  
R Region ◽  
Hiv 1

Although two strand transfer events are indispensable for the synthesis of double-stranded DNA and establishing HIV-1 infection, the molecular basis of these phenomena is still unclear. The first obligatory template switching event occurs just at the beginning of the virus replication cycle and involves two copies of the 97-nucleotide long R region, located one each at the both ends of the HIV-1 genome (HIV-1 R). Thus, one can expect that the molecular mechanism of this process is similar to the mechanism of homologous recombination which operates in RNA viruses. To verify the above-mentioned hypothesis, we attempted to assess the recombination activity of HIV-1 R. To this end, we tested in vitro, how effectively it induces template switching by HIV-1 RT in comparison with another well-characterized sequence supporting frequent homologous crossovers in an unrelated virus (R region derived from Brome mosaic virus--BMV R). We also examined if the RNA sequences neighboring HIV-1 R influence its recombination activity. Finally, we tested if HIV-1 R could cause BMV polymerase complex to switch between RNA templates in vivo. Overall, our results have revealed a relatively low recombination activity of HIV-1 R as compared to BMV R. This observation suggests that different factors modulate the efficiency of the first obligatory strand transfer in HIV-1 and the homology-driven recombination in RNA viruses.

scholarly journals Chikungunya Virus Fidelity Variants Exhibit Differential Attenuation and Population Diversity in Cell Culture and Adult Mice

2018 ◽  
Vol 93 (3) ◽  
Author(s):  
Kasen K. Riemersma ◽  
Cody Steiner ◽  
Anil Singapuri ◽  
Lark L. Coffey
Keyword(s):  
Global Health ◽  
Chikungunya Virus ◽  
Rna Viruses ◽  
Population Diversity ◽  
Mutation Rates ◽  
High Fidelity ◽  
Health Threat ◽  
Arthritic Disease ◽  
Adult Mice

ABSTRACTChikungunya virus (CHIKV) is a reemerging global health threat that produces debilitating arthritis in people. Like other RNA viruses with high mutation rates, CHIKV produces populations of genetically diverse genomes within a host. While several known CHIKV mutations influence disease severity in vertebrates and transmission by mosquitoes, the role of intrahost diversity in chikungunya arthritic disease has not been studied. In this study, high- and low-fidelity CHIKV variants, previously characterized by alteredin vitropopulation mutation frequencies, were used to evaluate how intrahost diversity influences clinical disease, CHIKV replication, and antibody neutralization in immunocompetent adult mice inoculated in the rear footpads. Both high- and low-fidelity mutations were hypothesized to attenuate CHIKV arthritic disease, replication, and neutralizing antibody levels compared to wild-type (WT) CHIKV. Unexpectedly, high-fidelity mutants elicited more severe arthritic disease than the WT despite comparable CHIKV replication, whereas a low-fidelity mutant produced attenuated disease and replication. Serum antibody developed against both high- and low-fidelity CHIKV exhibited reduced neutralization of WT CHIKV. Using next-generation sequencing (NGS), the high-fidelity mutations were demonstrated to be genetically stable but produced more genetically diverse populations than WT CHIKV in mice. This enhanced diversification was subsequently reproduced after serialin vitropassage. The NGS results contrast with previously reported population diversities for fidelity variants, which focused mainly on part of the E1 gene, and highlight the need for direct measurements of mutation rates to clarify CHIKV fidelity phenotypes.IMPORTANCECHIKV is a reemerging global health threat that elicits debilitating arthritis in humans. There are currently no commercially available CHIKV vaccines. Like other RNA viruses, CHIKV has a high mutation rate and is capable of rapid intrahost diversification during an infection. In other RNA viruses, virus population diversity associates with disease progression; however, potential impacts of intrahost viral diversity on CHIKV arthritic disease have not been studied. Using previously characterized CHIKV fidelity variants, we addressed whether CHIKV population diversity influences the severity of arthritis and host antibody response in an arthritic mouse model. Our findings show that CHIKV populations with greater genetic diversity can cause more severe disease and stimulate antibody responses with reduced neutralization of low-diversity virus populationsin vitro. The discordant high-fidelity phenotypes in this study highlight the complexity of inferring replication fidelity indirectly from population diversity.

scholarly journals Modelling and in vitro testing of the HIV-1 Nef fitness landscape

2019 ◽  
Vol 5 (2) ◽  
Author(s):  
John P Barton ◽  
Erasha Rajkoomar ◽  
Jaclyn K Mann ◽  
Dariusz K Murakowski ◽  
Mako Toyoda ◽  
...  
Keyword(s):  
Clinical Data ◽  
Fitness Landscape ◽  
Effective Vaccine ◽  
In Vitro Testing ◽  
Multifunctional Protein ◽  
Model Predictions ◽  
The Cost ◽  
Hiv 1

Abstract An effective vaccine is urgently required to curb the HIV-1 epidemic. We have previously described an approach to model the fitness landscape of several HIV-1 proteins, and have validated the results against experimental and clinical data. The fitness landscape may be used to identify mutation patterns harmful to virus viability, and consequently inform the design of immunogens that can target such regions for immunological control. Here we apply such an analysis and complementary experiments to HIV-1 Nef, a multifunctional protein which plays a key role in HIV-1 pathogenesis. We measured Nef-driven replication capacities as well as Nef-mediated CD4 and HLA-I down-modulation capacities of thirty-two different Nef mutants, and tested model predictions against these results. Furthermore, we evaluated the models using 448 patient-derived Nef sequences for which several Nef activities were previously measured. Model predictions correlated significantly with Nef-driven replication and CD4 down-modulation capacities, but not HLA-I down-modulation capacities, of the various Nef mutants. Similarly, in our analysis of patient-derived Nef sequences, CD4 down-modulation capacity correlated the most significantly with model predictions, suggesting that of the tested Nef functions, this is the most important in vivo. Overall, our results highlight how the fitness landscape inferred from patient-derived sequences captures, at least in part, the in vivo functional effects of mutations to Nef. However, the correlation between predictions of the fitness landscape and measured parameters of Nef function is not as accurate as the correlation observed in past studies for other proteins. This may be because of the additional complexity associated with inferring the cost of mutations on the diverse functions of Nef.

scholarly journals A viability qPCR dilemma: Are longer amplicons better?

2020 ◽  
Author(s):  
Wannes Van Holm ◽  
Justien Ghesquière ◽  
Nico Boon ◽  
Tim Verspecht ◽  
Kristel Bernaerts ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Limiting Factors ◽  
Accurate Assessment ◽  
Trade Off ◽  
Qpcr Data ◽  
Amplicon Length ◽  
Wide Range ◽  
Length Range ◽  
The Cost ◽  
Micro Organisms

The development of viability qPCR (v-qPCR) has allowed for a more accurate assessment of the viability of a microbial sample by limiting the amplification of DNA from dead cells. Although valuable, v-qPCR is not infallible. One of the most limiting factors for accurate live/dead distinction is the length of the qPCR amplicon used. However, no consensus or guidelines exist for selecting and designing amplicon lengths for optimal results. In this study, a wide range of incrementally increasing amplicon lengths (68-906 bp) was used on live and killed cells of nine bacterial species treated with viability dye (PMA). Increasing amplicon lengths up to approximately 200 bp resulted in increasing quantification cycle (Cq) differences between live and killed cells, while maintaining a good qPCR efficiency. Longer amplicon lengths, up to approximately 400 bp, further increased Cq difference, but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in Cq differences was observed. Importance Viability qPCR (v-qPCR) has evolved to a valuable, mainstream technique for determining the number of viable micro-organisms in samples by qPCR. Amplicon length is known to be positively correlated with the ability to distinguish between live and dead bacteria but is negatively correlated with qPCR efficiency. This trade-off is often not taken into account and might have an impact on the accuracy of v-qPCR data. Currently there is no consensus on the optimal amplicon length. This paper provides methods to determine the optimal amplicon length and suggests an amplicon length range for optimal v-qPCR, taking into consideration the trade-off between qPCR efficiency and live-dead distinction.

scholarly journals Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido-2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine.

1989 ◽  
Vol 169 (3) ◽  
pp. 933-951 ◽  
Author(s):  
C F Perno ◽  
R Yarchoan ◽  
D A Cooney ◽  
N R Hartman ◽  
D S Webb ◽  
...  
Keyword(s):  
Antiviral Effect ◽  
Productive Infection ◽  
Biochemical Basis ◽  
Gm Csf ◽  
The Face ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Anti Hiv ◽  
Hiv 1

We have investigated the influence of granulocyte-macrophage CSF (GM-CSF) on the replication of HIV-1 in cells of monocyte/macrophage (M/M) lineage, and its effect on the anti-HIV activity of several 2'3'-dideoxynucleoside congeners of thymidine in these cells in vitro. We found that replication of both HTLV-IIIBa-L (a monocytotropic strain of HIV-1) and HTLV-IIIB (a lymphocytotropic strain) is markedly enhanced in M/M, but not in lymphocytes exposed to GM-CSF in culture. Moreover, GM-CSF reduced the dose of HIV required to obtain productive infection in M/M. Even in the face of this increased infection, GM-CSF also enhanced the net anti-HIV activity of 3'-azido-2'3'-dideoxythymidine (AZT) and several related congeners: 2'3'-dideoxythymidine (ddT), 2'3'-dideoxy-2'3'-didehydrothymidine (D4T), and 3'-azido-2'3'-dideoxyuridine (AZddU). Inhibition of viral replication in GM-CSF-exposed M/M was achieved with concentrations of AZT and related drugs, which were 10-100 times lower than those inhibitory for HIV-1 in monocytes in the absence of GM-CSF. Other dideoxynucleosides not related to AZT showed unchanged or decreased anti-HIV activity in GM-CSF-exposed M/M. To investigate the possible biochemical basis for these effects, we evaluated the metabolism of several drugs in M/M exposed to GM-CSF. We observed in these cells markedly increased levels of both parent and mono-, di-, and triphosphate anabolites of AZT and D4T compared with M/M not exposed to GM-CSF. By contrast, only limited increases of endogenous competing 2'-deoxynucleoside-5'-triphosphate pools were observed after GM-CSF exposure. Thus, the ratio of AZT-5'-triphosphate/2'-deoxythymidine-5'-triphosphate and 2'3'-dideoxy-2'3'-didehydrothymidine-5'-triphosphate/2'-deoxythymi dine- 5'-triphosphate is several-fold higher in GM-CSF-exposed M/M, and this may account for the enhanced activity of such drugs in these cells. Taken together, these findings suggest that GM-CSF increases HIV-1 replication in M/M, while at the same time enhancing the anti-HIV activity of AZT and related congeners in these cells. These results may have implications in exploring new therapeutic strategies in patients with severe HIV infection.

scholarly journals Spontaneous Mutations in HIV-1 Gag, protease, RT p66 in the first replication cycle and how they appear: Insights from an in vitro BSL2 assay on mutation rates and types

2019 ◽  
Author(s):  
Joshua Yi Yeo ◽  
Darius Wen-Shuo Koh ◽  
Ping Yap ◽  
Ghin-Ray Goh ◽  
Samuel Ken-En Gan
Keyword(s):  
Codon Usage ◽  
Mutation Rates ◽  
Free Energies ◽  
Host Proteins ◽  
Free System ◽  
Adenosine Deaminases ◽  
Functional Sites ◽  
Specific Effects ◽  
Hiv 1

AbstractWhile drug resistant mutations in HIV-1 is largely credited to its error prone HIV-1 RT, host proteins such as deaminases may also play a role generating mutations. Many HIV-1 RT mutational in vitro studies utilize reporter genes (LacZ) as template, leaving out the possible contribution of HIV codon usage and gene-specific effects. To address this gap, we studied HIV-1 RT mutation rates and bias on its own Gag, protease, and RT p66 genes in an in-vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease and RT p66 at 4.71 x 10−5, 6.03 x 10−5, and 7.09 x 10−5 mutations/bp respectively. Gag and p66 genes showed a large number of ‘A to G’ hypermutations likely due to cellular adenosine deaminases. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 x 10−4 mutations/bp) and that ‘A to G’ mutations occurred in regions reminiscent of ADAR neighbour preferences. Mutational free energies by the ‘A to G’ mutations revealed an avoidance of destabilizing effects with the natural p66 gene codon usage providing barriers to ADAR effects. Our study demonstrates the importance of studying mutation emergence in HIV genes to understand how fast drug resistance can emerge, sometimes with contributions of host deaminases, providing transferable applications to how new viral diseases and drug resistances can emerge.

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