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. 2012 Oct;9(10):1266-74.
doi: 10.4161/rna.22081. Epub 2012 Sep 20.

Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus

Andrey Chursov  1 , Sebastian J Kopetzky, Ignaty Leshchiner, Ivan Kondofersky, Fabian J Theis, Dmitrij Frishman, Alexander Shneider
Affiliations

Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus

Andrey Chursov et al. RNA Biol. 2012 Oct.

Abstract

For decades, cold-adapted, temperature-sensitive (ca/ts) strains of influenza A virus have been used as live attenuated vaccines. Due to their great public health importance it is crucial to understand the molecular mechanism(s) of cold adaptation and temperature sensitivity that are currently unknown. For instance, secondary RNA structures play important roles in influenza biology. Thus, we hypothesized that a relatively minor change in temperature (32-39°C) can lead to perturbations in influenza RNA structures and, that these structural perturbations may be different for mRNAs of the wild type (wt) and ca/ts strains. To test this hypothesis, we developed a novel in silico method that enables assessing whether two related RNA molecules would undergo (dis)similar structural perturbations upon temperature change. The proposed method allows identifying those areas within an RNA chain where dissimilarities of RNA secondary structures at two different temperatures are particularly pronounced, without knowing particular RNA shapes at either temperature. We identified such areas in the NS2, PA, PB2 and NP mRNAs. However, these areas are not identical for the wt and ca/ts mutants. Differences in temperature-induced structural changes of wt and ca/ts mRNA structures may constitute a yet unappreciated molecular mechanism of the cold adaptation/temperature sensitivity phenomena.

Keywords: RNA; influenza; structure; temperature; vaccine.

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Figures

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Figure 1. Comparison of significantly changing positions between the PA mRNA of Len/wt (upper 7 rows) and Len/17/ca (lower 7 rows). Each row corresponds to a difference vector v32–33, …, v32–39 containing changes of base pairing probabilities between 32°C and a particular higher temperature. Positions in which base paring probabilities significantly change with temperature elevation in both sequences and those where these changes only affect one of the phenotypes are marked blue and orange, respectively. Only the first 40 bases of each sequence are shown; position numbers of the coding sequence are indicated at the top of the alignment.
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Figure 2. Distributions of significantly changing positions along the PB2 mRNAs of Arb/wt and Arb/ca. A sliding window of size 20 was moved in steps of 1 position over the vector v32–39 and the percentage of significantly changing positions in the window was calculated for each possible starting position. The resulting density plots are depicted in Figure 2A and Figure 2C. Location of clusters of significantly changing positions identified by the DBSCAN algorithm are depicted in Figure 2B and Figure 2D with gray color. Synonymous and non-synonymous mutations are depicted in Figure 2B and Figure 2D with red and blue vertical lines, respectively.
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Figure 3. The histogram of differences of the probability values of nucleotides to be in a double-stranded conformation for PB1 Arb/wt upon temperature change between 32°C and 39°C. The vector of probabilities for 32°C was subtracted from the vector for 39°C.
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Figure 4. Density of significantly changing positions in determined clusters vs. length. (A) Clusters that occur only in wt mRNAs but are not statistically significant (37). (B) Clusters occurring in both wt and ca/ts mRNAs (126). (C) Clusters that occur only in ca/ts mutants but are not statistically significant (45). (D) Statistically significant clusters (9 of them occur only in ca/ts mutants and 1 of them occurs only in wt mRNA). Different colors show different numbers of clusters that have identical values of length and density. Black, one cluster; red, two clusters; green, three clusters; blue, four clusters.
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