Comparative mutational analyses of influenza A viruses

Abstract

The error-prone RNA-dependent RNA polymerase (RdRP) and external selective pressures are the driving forces for RNA viral diversity. When confounded by selective pressures, it is difficult to assess if influenza A viruses (IAV) that have a wide host range possess comparable or distinct spontaneous mutational frequency in their RdRPs. We used in-depth bioinformatics analyses to assess the spontaneous mutational frequencies of two RdRPs derived from human seasonal (A/Wuhan/359/95; Wuhan) and H5N1 (A/Vietnam/1203/04; VN1203) viruses using the mini-genome system with a common firefly luciferase reporter serving as the template. High-fidelity reverse transcriptase was applied to generate high-quality mutational spectra which allowed us to assess and compare the mutational frequencies and mutable motifs along a target sequence of the two RdRPs of two different subtypes. We observed correlated mutational spectra (τ correlation P < 0.0001), comparable mutational frequencies (H3N2:5.8 ± 0.9; H5N1:6.0 ± 0.5), and discovered a highly mutable motif "(A)AAG" for both Wuhan and VN1203 RdRPs. Results were then confirmed with two recombinant A/Puerto Rico/8/34 (PR8) viruses that possess RdRP derived from Wuhan or VN1203 (RG-PR8×Wuhan(PB2, PB1, PA, NP) and RG-PR8×VN1203(PB2, PB1, PA, NP)). Applying novel bioinformatics analysis on influenza mutational spectra, we provide a platform for a comprehensive analysis of the spontaneous mutation spectra for an RNA virus.

Keywords: H5N1; bioinformatics; influenza A virus; mutational spectra; polymerase fidelity.

FIGURE 1.

Mini-genome system for assessing the intrinsic influenza polymerase mutation rate and mutational spectra. A mini-genome system was applied to assess the RdRP activity in human 293T cells using the reporter plasmid driven by human Polymerase I promoter encoding firefly luciferase flanked by the 3′- and 5′-noncoding regions of influenza segment seven. Human 293T cells were cotransfected with the PB2, PB1, PA, NP, and the firefly luciferase and the cells were lysed for total RNA isolation or for luciferase activity measurements at 24 h post-transfection. The negative control transfection contained no IAV PB1 plasmid. (A) PCR amplification of the luciferase reporter gene driven by the RdRPs derived from Wuhan (Wu) or VN1203 (VN) viruses. Transfections were performed in triplicates and the negative controls (without PB1) were included in parallel. (B) Real-time PCR assay for firefly luciferase mRNA quantification and firefly luciferase activity as a proximate for the polymerase activity of Wuhan and VN1203 RdRPs. The results shown (mean ± SD) were from triplicated wells in one out of two independently performed experiments.

FIGURE 2.

The two different RdRPs show comparable transition mutational bias in with the (A) polymerase-driven spectra and (B) live virus spectra. (A) Mutational preference of A/Wuhan/359/95 and A/Vietnam/1203/04 polymerase complexes. (B) Mutational preference of recombinant RG-PR8×Wuhan^{PB2, PB1, PA, NP} and RG-PR8×VN1203^{PB2, PB1, PA, NP} viruses using the common HA gene as the template. The percentages (%) depict the specific nucleotide in yellow that mutate to the nucleotide in red, among the total number of nucleotide substitutions. Sequences generated from two independent experiments are pooled for the analysis.