Escherichia coli DNA polymerase III ε subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT–PCR procedures

Arezi, Bahram; Hogrefe, Holly H.

doi:10.1016/j.ab.2006.10.009

Cited by 50 publications

(36 citation statements)

References 17 publications

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“…The assay background levels of substitutions for plasmid controls were significantly lower with only 67 mutants observed among 3,764,286 tested baseread positions. The reverse transcription step is associated with an error rate of 1.6 × 10 −5 (displaying a 5-fold preference for transitions over transversions) (23), which is also significantly lower when compared with our observed error rates of 9.4 × 10 −3 per patient.…”

Section: Resultsmentioning

confidence: 44%

“…RNA was also isolated from 10 6 1a H77-Rluc replicon cells (31) using the RNeasy minikit (Qiagen). To reduce the contribution of errors introduced during the reverse transcription step, we utilized the high fidelity AccuScript RT (Stratagene) (23). The NS3 protease region was amplified as two overlapping amplicons using genotype specific barcoded primers.…”

Section: Methodsmentioning

confidence: 99%

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Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Powdrill

Tchesnokov²,

Kozak³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

133

104

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The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10 −3 per site) for G∶U∕U∶G mismatches. The strong preference for G∶U∕U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naïve patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.A pproximately 170 million people worldwide are infected with hepatitis C virus (HCV) (1). Chronically infected individuals are at risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma (2). Although the infection can be cured with a combination of interferon-α and ribavirin, severe side effects and complicated treatment schedules can compromise therapy (3). Moreover, the response is particularly poor in patients infected with HCV genotype 1, which is the most prevalent genotype in North America and Europe (4).Several direct-acting antivirals (DAAs) targeting important viral and host factors are currently undergoing clinical evaluation. Drugs acting against the viral NS5B RNA-dependent RNA polymerase (RdRp) or NS3 protease are in advanced clinical trials, with the protease inhibitors (PIs) telaprevir and boceprevir recently gaining FDA approval. Combining interferon-α and ribavirin with either PI can produce increases in sustained virological response when compared with the combination of interferon-α and ribavirin without PI (5, 6). However, the emergence of resistance-conferring mutations can lead to treatment failure (7). Like other RNA viruses, HCV shows quasispecies-like characteristics (8). Accordingly, resistant variants are selected from a genetically highly diverse population. Resistance to PIs is rapidly selected in cell-based replicon systems and in vivo (9). Similar observations have been made with the various classes of nonnucleoside analogue inhibitors (NNIs) of NS5B (9). Moreover, mutations that decrease susceptibility to highly potent inhibitors of the RNA binding protein NS5A also emerge rapidly in the replicon system (10). In contrast, the barrier to the development of resistance to nucleoside analogue inhibitors (NIs) appears to be significantly higher. The 2′-C-methylated NIs were shown to select in v...

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Section: Resultsmentioning

confidence: 44%

Section: Methodsmentioning

confidence: 99%

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Powdrill

Tchesnokov²,

Kozak³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

133

104

View full text Add to dashboard Cite

show abstract

“…These results are in good agreement with the two-to fourfold lower error rates of the group II intron RTs in the M13-based lacZ forward mutation assay, where the fidelity of the enzymes was measured on a single RNA template (see above). In both assays, the error rates measured for the TeI4c and GsI-IIC group II intron RTs are lower than those reported in the literature for retroviral RTs (M-MLV RT; 3.6-6.7 × 10 −5 ; HIV-1, >10 −4 ) (Ji and Loeb 1992;Potter et al 2003;Arezi and Hogrefe 2007).…”

Section: Next-generation Sequencing Of Human Cdna Librariesmentioning

confidence: 57%

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Mohr

Ghanem

Smith

et al. 2013

RNA

187

301

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Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3 ′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

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“…Henceforth, we may expect $34 mutations in our sample to be due to errors during PCR. Unfortunately, this is not the only source of error; the error rate of MMLV RT is around 3.3 3 10 À5 m/b/r (Arezi and Hogrefe 2007), which means that we may expect as well $24 mutations to be produced during retrotranscription. Since we have obtained 52 mutations, someone may argue that all of them must result from errors during either retrotranscription or PCR amplification.…”

Section: Resultsmentioning

confidence: 99%

The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

Tromas

Elena

2010

Genetics

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Knowing mutation rates and the molecular spectrum of spontaneous mutations is important to understanding how the genetic composition of viral populations evolves. Previous studies have shown that the rate of spontaneous mutations for RNA viruses widely varies between 0.01 and 2 mutations per genome and generation, with plant RNA viruses always occupying the lower side of this range. However, this peculiarity of plant RNA viruses is based on a very limited number of studies. Here we analyze the spontaneous mutational spectrum and the mutation rate of Tobacco etch potyvirus, a model system of positive sense RNA viruses. Our experimental setup minimizes the action of purifying selection on the mutational spectrum, thus giving a picture of what types of mutations are produced by the viral replicase. As expected for a neutral target, we found that transitions and nonsynonymous (including a few stop codons and small deletions) mutations were the most abundant type. This spectrum was notably different from the one previously described for another plant virus. We have estimated that the spontaneous mutation rate for this virus was in the range 10 À6 À10À5 mutations per site and generation. Our estimates are in the same biological ballpark that previous values reported for plant RNA viruses. This finding gives further support to the idea that plant RNA viruses may have lower mutation rates than their animal counterparts.T HE rate of spontaneous mutation is a key parameter to understanding the genetic structure of populations over time. Mutation represents the primary source of genetic variation on which natural selection and genetic drift operate. Although the exact value of the mutation rate is important for several evolutionary theories, accurate estimates are available only for a handful of organisms. RNA viruses show mutation rates that are orders of magnitude higher than those of their DNA-based hosts and in the range of 0

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Escherichia coli DNA polymerase III ε subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT–PCR procedures

Cited by 50 publications

References 17 publications

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

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