Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection

Abstract

Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.DOI: http://dx.doi.org/10.7554/eLife.12735.001

Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species

“…In addition, the dinucleotide frequency has been shown to change following cross-species transmission events, such as the marked decrease in CpG frequency as influenza A virus jumps from birds to humans (20,21). Studies of influenza viruses and picornaviruses indicate that the innate immune response might recognize RNA-specific CpG motifs, such that the suppression of CpG in viruses could assist immune evasion (22)(23)(24). In contrast, other studies have suggested that dinucleotide bias in viruses simply reflects background mutation pressure.…”

Sequence-Specific Sensing of Nucleic Acids

“…However, experimental use of kinase inhibitors reverted the attenuation phenotype of the mutants, suggesting a role for an unknown PRR in mediating a dinucleotide usage-specific immune response [24]. In a follow-up study on influenza virus, the authors demonstrated that, despite marked attenuation of replication, virus mutants enriched in CpG and UpA induced higher inflammatory cytokines and adaptive responses in vivo [25].…”

“…IN: Integrase. Table 1 Interactions between viral sequence patterns and innate immune pathways deciphered by large-scale synonymous recoding of viral genomes Virus Sequence pattern studied Innate immune pathway involved Reference Influenza (A/U)CG(A/U) IFN-I secretion, TLR7-dependent [23] Simian Immunodeficiency Virus Nucleotide bias IFN-I, IRF3-dependent [28] Echovirus CpG and UpA Unknown PRR, IRF3-independent [24] Infuenza CpG and UpA Pro-inflammatory cytokines Unknown PRR [25].…”

Attenuation of Viruses by Large-Scale Recoding of their Genomes: the Selection Is Always Biased

“…It was discovered that codon and codon pair deoptimization of vertebrate viruses not only increases the number of codons, or codon pairs that are underrepresented in coding sequences of the host, but also increases the frequency of CpG and, to lesser degree, TpA (UpA) dinucleotides in recoded sequences [45,54,55,58]. The increase of CpG and TpA dinucleotides by codon pair deoptimization is inadvertent, as codon pairs that contain CpG and TpA dinucleotides at the codon pair boundary (NNC-p-GNN) are among the most underrepresented codon pairs in vertebrates [45,55].…”

“…A subsequent study from the same group demonstrated that elevation of CpG frequencies in influenza A virus can also result in moderate attenuation of the virus in vitro and in vivo [58]. However, since recoded viruses with increased CpG or UpA frequencies did not have the same codon bias as the control-unmodified and permuted-viruses, changes in the codon bias could confound observed viral properties.…”