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Viral Mutations00:36

Viral Mutations

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A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material...
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Single Nucleotide Polymorphisms-SNPs01:05

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A single nucleotide polymorphism or SNP is a single nucleotide variation at a specific genomic position in a large population. It is the most prevalent type of sequence variation found in the human genome. Point mutations that occur in more than 1% of the population qualify as SNPs. These are present once every 1000 nucleotides on an average in the human genome. Replacement of a purine with another purine (A/G) or a pyrimidine with another pyrimidine (C/T) is known as a transition. In contrast,...
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Mutations in Microorganisms01:18

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Mutations are heritable changes in an organism’s genome involving alterations in the base sequence of DNA or RNA. These changes can influence cellular processes and phenotypic traits, potentially transforming the unaltered wild type into a mutant form. Such changes, termed forward mutations, are pivotal in shaping the genetic diversity of organisms.RNA viruses exhibit the highest mutation rates due to the absence of robust proofreading mechanisms during genome replication. In contrast,...
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Mutations01:35

Mutations

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Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
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Mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity.

Everett Clinton Smith1, James Brett Case2, Hervé Blanc3

  • 1Department of Pediatrics, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA The Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA.

Journal of Virology
|April 10, 2015
PubMed
Summary
This summary is machine-generated.

The nonstructural protein 10 (nsp10) of coronaviruses is crucial for maintaining high-fidelity replication. Disrupting the nsp10-nsp14 interaction in murine hepatitis virus reduces replication fidelity, making it more sensitive to RNA mutagens.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Coronaviruses (CoVs) possess a unique 3'→5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) essential for high-fidelity RNA replication.
  • The nsp14-ExoN enzyme functions in conjunction with the RNA-dependent RNA polymerase (nsp12-RdRp) and is enhanced by the nonenzymatic protein nsp10.
  • The precise role of nsp10 in regulating CoV replication fidelity remained uncharacterized.

Purpose of the Study:

  • To investigate the function of nsp10 in the regulation of coronavirus replication fidelity.
  • To determine if nsp10 modulates the activity of the nsp14-ExoN proofreading mechanism.
  • To identify viral protein determinants crucial for maintaining the genetic integrity of CoV genomes.

Main Methods:

  • Engineered single and double alanine substitution mutations in the nsp10 gene of murine hepatitis virus (MHV-A59) to disrupt the nsp10-nsp14 interaction.
  • Assessed the replication fidelity of mutant viruses by measuring sensitivity to RNA mutagens like 5-fluorouracil (5-FU), 5-azacytidine, and ribavirin.
  • Compared viral RNA synthesis kinetics and peak titers between wild-type and mutant viruses.

Main Results:

  • A specific nsp10 mutant (R80A/E82A) exhibited a five- to tenfold increase in sensitivity to 5-FU, indicating reduced replication fidelity.
  • This decreased fidelity phenotype was confirmed with other nucleoside analogs and was dependent on the presence of nsp14-ExoN activity.
  • The nsp10 mutant demonstrated comparable RNA synthesis kinetics and peak titers to wild-type virus, suggesting fidelity is the primary affected parameter.

Conclusions:

  • The nonenzymatic protein nsp10 plays a significant role in maintaining coronavirus replication fidelity.
  • These findings support the hypothesis that nsp10 regulates the proofreading activity of nsp14-ExoN during viral replication.
  • Coronaviruses employ multiple proteins, including nsp10, to ensure the integrity of their large RNA genomes.