Stability-mediated epistasis constrains the evolution of an influenza protein

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Stability-mediated epistasis constrains the evolution of an influenza protein

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DOI: http://dx.doi.org/10.7554/eLife.00631Published May 14, 2013Cite as eLife 2013;2:e00631
  • Figure 1.
    Download figureOpen in new tabDownload powerpointFigure 1. Outline of experiment designed to parallel Maynard Smith’s analogy.

    The actual evolutionary trajectory involves the accumulation of mutations, and consists of a series of evolutionary intermediates. We recreate and experimentally assay each of these evolutionary intermediates. We also introduce each mutation individually into the original parent sequence, and experimentally assay these single mutants. If Maynard Smith is correct, each of the naturally occurring evolutionary intermediates should be a functional protein. However, some of the single mutants could exhibit impaired function if there is significant epistasis among mutations along the evolutionary trajectory.

    DOI: http://dx.doi.org/10.7554/eLife.00631.003

  • Figure 4.
    Download figureOpen in new tabDownload powerpointFigure 4. Effects of the individually deleterious mutations in the evolutionary intermediates in which they occurred.

    (A) L259S impairs the transcriptional activity and viral growth of both the parent Aichi/1968 and the evolutionary intermediate Step 10, but is rescued by N334H in both backgrounds. N334H alone has little effect on activity or growth in either background. The actual evolutionary trajectory involved the fixation of L259S and N334H in an unknown order. (B) R384G impairs activity and ablates growth of Aichi/1968, but has no effect on activity and a reduced adverse effect on growth in the high-confidence evolutionary intermediate (Step 21) in which it and several other mutations occurred in an unknown order. Addition of E375G to Step 21 with R384G fully rescues viral growth, but E375G alone worsens the impact of R384G. The reversion of L259S that preceded Step 21 plays an important role in enabling R384G, as the evolutionary intermediate without this reversion (Step 20) is more negatively affected by R384G. (C) V280A is deleterious in Aichi/1968 but not in the Step 35 evolutionary intermediate in which it actually occurred. M136I, which precedes V280A, largely rescues its effect.

    DOI: http://dx.doi.org/10.7554/eLife.00631.015

Supplementary file 1.The full vRNAs in the reverse-genetics plasmids used in this study.

A text file giving the vRNAs inserted between the RNA polymerase I promoter and terminator in the reverse-genetics plasmids pHWAichi68-NP, pHWNan95-PB2, pHWNan95-PB1, pHWNan95-PA, pHWAichi68-PB2, pHWAichi68-PB1, pHWAichi68-PA, pHWBR07-PB2, pHWBR07-PB1, pHWBR07-PA, pHW184-HA, pHW186-NA, pHW187-M, pHW188-NS, and pHH-PB1flank-eGFP.

DOI: http://dx.doi.org/10.7554/eLife.00631.038

Download source data [supplementary-file-1.media-10.txt]
Supplementary file 2.The protein-coding sequences of all NP variants used in this study.

A text file giving the coding sequences for the NP variants used in this study.

DOI: http://dx.doi.org/10.7554/eLife.00631.039

Download source data [supplementary-file-2.media-11.txt]
Figure 2—source code 1.The sequence data and source code used to generate the evolutionary trajectory and phylogenetic tree.

This code is in the form of a ZIP file with BEAST input files and Python code. A README file explains the contents.

DOI: http://dx.doi.org/10.7554/eLife.00631.005

Download source data [figure-2—source-code-1.media-1.zip]
Figure 3—source data 1.Summary of transcriptional activity data (mean and standard error) for all variants from this study.

The means and standard errors are computed from at least triplicate assays, and are reported standardized so the value for Aichi/1968 NP is at 1. The data are provided in a CSV file.

DOI: http://dx.doi.org/10.7554/eLife.00631.009

Download source data [figure-3—source-data-1.media-2.csv]
Figure 3—source data 2.Summary of viral growth data (mean and standard error) for all variants for which this property was measured in this study.

The means and standard errors are computed from at least triplicate assays, and are reported standardized so the value for Aichi/1968 NP is at 1. The data are provided in a CSV file.

DOI: http://dx.doi.org/10.7554/eLife.00631.010

Download source data [figure-3—source-data-2.media-3.csv]
Figure 3—source data 3.Transcriptional activity data in the alternative polymerase genetic backgrounds shown in Figure 3—figure supplement 3.

The data are provided in a CSV file.

DOI: http://dx.doi.org/10.7554/eLife.00631.011

Download source data [figure-3—source-data-3.media-4.csv]
Figure 6—source data 1.A table of all of the melting temperatures and the changes in stability relative to Aichi/1968, in CSV format.

DOI: http://dx.doi.org/10.7554/eLife.00631.019

Download source data [figure-6—source-data-1.media-5.csv]
Figure 7—source data 1.The activity, viral growth, and stability data shown in Figure 7.

The data are provided in CSV format.

DOI: http://dx.doi.org/10.7554/eLife.00631.029

Download source data [figure-7—source-data-1.media-6.csv]
Figure 8—source data 1.Literature-characterized NP human CTL epitopes that include residues 259, 280, or 384 and contain sequences conserved or nearly conserved in Aichi/1968.

The epitopes are listed in a TXT file.

DOI: http://dx.doi.org/10.7554/eLife.00631.032

Download source data [figure-8—source-data-1.media-7.txt]
Figure 8—source code 1.The input data files and the custom Python scripts used to identify human CTL epitopes in NP.

The code and data are provided as a ZIP file, which contains a README text file that describes the contents in more detail.

DOI: http://dx.doi.org/10.7554/eLife.00631.033

Download source data [figure-8—source-code-1.media-8.zip]
Figure 8—source code 2.The data and source code used for the dN/dS analysis.

The code and data are provided in a ZIP file; a README file explains the contents.

DOI: http://dx.doi.org/10.7554/eLife.00631.034

Download source data [figure-8—source-code-2.media-9.zip]