A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses
- University of Michigan, United States
Figures
Figure 1
Influenza mutation rates by PrimerID next generation sequencing.
(A) Segment 3 (PA) RNA was isolated from either cells transfected with pol I expression plasmids or cell-free supernatants of cells infected with influenza virus. These RNA were reverse transcribed …
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Figure 1—source data 1
Spreadsheet with frequencies of mutations to stop codons in plasmid- and virus-derived RNA.
- https://doi.org/10.7554/eLife.26437.003
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Figure 1—source data 2
Spreadsheet with mutation frequency by class for plasmid- and virus-derived RNA.
- https://doi.org/10.7554/eLife.26437.004
Figure 2
Characterization of mutant ΔHA-GFP influenza viruses.
(A) Fluorescent images of cells infected with mutant ΔHA-GFP (shown are data for the A to C virus, see Table 1) and stained with Hoechst and anti-GFP Alexa 647 conjugate. Cells were imaged at 4x …
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Figure 2—source data 1
Spreadsheet with virus titer (GFP+ virus per ml) in imaging plate at the indicated time points and temperatures.
- https://doi.org/10.7554/eLife.26437.007
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Figure 2—source data 2
Spreadsheet with replicate fitness values for wild type and ΔHA-GFP viruses as shown in Figure 2C.
- https://doi.org/10.7554/eLife.26437.008
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Figure 2—source data 3
Spreadsheet with minimum free energy of RNA folding by window start position as shown in Figure 2D.
- https://doi.org/10.7554/eLife.26437.009
Figure 3
Fluorescent Luria-Delbruck fluctuation test.
(A) General workflow for measuring the mutation rate using mutant ΔHA-GFP viruses. Parallel cultures of MDCK-HA cells were infected with passage one stocks of mutant ΔHA-GFP viruses at low …
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Figure 3—source data 1
Mutation rates for A to G, G to A, and G to C viruses in the presence and absence of AzaC, 5FU and Riba as measured by fluctuation test.
- https://doi.org/10.7554/eLife.26437.011
Figure 4
The mutation rates of influenza viruses replicated at 37°C.
(A) Measurements of A/Puerto Rico/8/1934 H1N1 viruses encoding the 12 different mutant ΔHA-GFP constructs. (B) Measurements of viruses encoding the replication complex (PB2, PB1, PA, and NP) from …
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Figure 4—source data 1
Mutation rates for all twelve mutational classes for PR8 and Hong Kong viruses as measured by fluctuation test.
- https://doi.org/10.7554/eLife.26437.013
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Figure 4—source data 2
Raw data for all experiments with Hong Kong viruses showing number of wells with n green cells (n = 0–10).
- https://doi.org/10.7554/eLife.26437.014
Figure 5
The effect of temperature on influenza A virus mutation rates.
Mutation rates were determined for A/Puerto Rico/8/1934 H1N1 viruses encoding the indicated mutant ΔHA-GFP constructs replicated at 32°C (blue), 37°C (black) and 39°C (red). Filled symbols represent …
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Figure 5—source data 1
Mutation rates for all twelve mutational classes for PR8 at the indicated temperatures as measured by fluctuation test.
- https://doi.org/10.7554/eLife.26437.016
Tables
Table 1
Non-fluorescent ΔHA-GFP constructs.
| Mutation Probed* | Nucleotide Sequece† | Amino acid Sequence‡ |
|---|---|---|
| WT eGFP | acc uac ggc | T Y G |
| A -> C | aAa uac ggc | K Y G |
| A -> G | acc uac gAc | T Y D |
| A -> U | acc Aac ggc | T N G |
| C -> A | acc uCc ggc | T S G |
| C -> G | acc uac gCc | T Y A |
| C -> U | acc Cac ggc | T H G |
| G -> A | acc uGc ggc | T C G |
| G -> C | uGg uac ggc§ | W Y G |
| G -> U | acc Gac ggc | T D G |
| U -> A | acc uUc ggc | T F G |
| U -> C | aUa uac ggc | I Y G |
| U -> G | acc uac gUc | T Y V |
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*Mutations are in the mRNA coding sense.
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†Nucleotides 193–201 of the eGFP reading frame are shown. Changes from wild type are in bold and italics. Site that allows reversion to fluorescence is capitalized.
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‡Amino acids 65–67 of eGFP are shown. Changes from wild type are in bold and italics.
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§This construct is able to revert to wild type GFP (S65).
Additional files
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Supplementary file 1
Nonsense mutation counts from PrimerID sequencing of the influenza PA gene.
- https://doi.org/10.7554/eLife.26437.017
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Supplementary file 2
Influenza A virus mutation rates for PR8 and Hong Kong viruses.
- https://doi.org/10.7554/eLife.26437.018
