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.

Stop-codon variations in non-structural protein NS1 of avian influenza viruses

“…A mutational analysis by Cheung et al, based on transiently expressed polymerase complexes from 2 distinct viruses in 293T cells, identified AAAG as a highly mutable sequence motif, and revealed that the viral polymerase favors transitions over transversions, with a predominance of A>G and U>C among the 4 possible transitions. 9 All these facts concur to a biased spectrum of mutations along the viral genome. Abdelwhab et al observed that by far the most prevalent C-terminal truncation occurs through conversion of codon 218 to a stop codon, thus resulting in the NS217 variant.…”

Influenza A Virus Cell Entry, Replication, Virion Assembly and Movement

“…Most studies on the influenza evolutionary process focus primarily on antigenic drift and antigenic shift. However, all the viral transcribed RNAs are subject to replication errors by the viral polymerase, which are estimated at 1 per 2,000–10,000 nucleotides ( 231 – 233 ). Consequently, both the viruses and the viral proteins are likely to exist as large heterogeneous populations during an infection.…”

Application of the New Generation of Sequencing Technologies for Evaluation of Genetic Consistency of Influenza A Vaccine Viruses

“…The segmented nature of the viral genome enables two viruses co-infecting the same cell to exchange their segments to produce reassortant progeny. Replication of influenza A viruses also results in mutated viral genomes because of the high error frequency of the RNA polymerase and actions by host defensive elements [3,4]. Mutations contribute to the emergence of new endemic strains and reassortment may lead to the emergence of epidemic or pandemic strains.…”

“…Several groups have isolated clones and used Sanger sequencing to identify mutations. Isolation of a sufficient number of clones has resulted in estimates of the mutation frequencies ranging from 6 × 10 −4 to 2 × 10 −6 [3,5,6]. Although the information about heterogeneity is of great interest, caution must be exercised to ensure that it is accurately reflected in sequence databases.…”