Large-scale sequence analysis reveals novel human-adaptive markers in PB2 segment of seasonal influenza A viruses

Abstract

To elucidate the adaptive strategies of influenza A viruses (IAVs) to human, we proposed a computational approach to identify human-adaptive mutations in seasonal IAVs, which have not been analyzed comprehensively. We compared representative PB2 sequences of 1425 avian IAVs and 2176 human IAVs and identified a total of 42 human-adaptive markers, including 28 and 31 markers in PB2 proteins of seasonal viruses H1N1 and H3N2, respectively. Notably, this comprehensive list encompasses almost all the markers identified in prior computational studies and 21 novel markers including an experimentally verified mutation K526R, suggesting the predictive power of our method. The strength of our analysis derives from the enormous amount of recently available sequences as well as the recognition that human-adaptive mutations are not necessarily conserved across subtypes. We also utilized mutual information to profile the inter-residue coevolution in PB2 protein. A total of 35 and 46 coevolving site pairs are identified in H1N1 and H3N2, respectively. Interestingly, 13 out of the 28 (46.4%) identified markers in H1N1 and 16 out of the 31 (51.6%) in H3N2 are embraced in the coevolving pairs. Many of them are paired with well-characterized human-adaptive mutations, indicating potential epistatic effect of these coevolving residues in human adaptation. Additionally, we reconstructed the PB2 evolutionary history of seasonal IAVs and demonstrated the distinct adaptive pathway of PB2 segment after reassortment from H1 to H3 lineage. Our study may provide clues for further experimental validation of human-adaptive mutations and shed light on the human adaptation process of seasonal IAVs.

Fig. 1. Host-specific signatures of seasonal IAVs in PB2 protein.

Localization of identified human-adaptive markers in the PB2 domains of seasonal IAVs H1N1 (a) and H3N2 (b). The cartoon representation of PB2 subunit was rendered by Pymol (PDB code: 4WSB). Domains are colored according to the color code in c. Experimentally verified markers are indicated in red sphere, computationally predicted markers are indicated in green, and novel markers are indicated in blue. c The positions of PB2 domains are color-coded and labeled according to their functions

Fig. 2. MI network of seasonal H1N1 IAV PB2 protein.

a Circular representation of PB2 protein and the MI network. The positions of PB2 domains are color-coded and labeled according to their functions. Arcs connect site pairs with normalized MI > 0.7. b Cytoscape representation of the MI network. Sites are represented as nodes. Edge between two nodes indicates the normalized MI > 0.7. Sites with experimentally verified human-adaptive mutations and computationally identified markers are highlighted in red and green, respectively, in a, b; blue nodes indicate novel markers

Fig. 3. MI network of seasonal H3N2 IAV PB2 protein.

a Circular representation of PB2 protein and the MI network. The positions of PB2 domains are color-coded and labeled according to their functions. Arcs connect site pairs with normalized MI > 0.7. b Cytoscape representation of the MI network. Sites are represented as nodes. Edge between two nodes indicates the normalized MI > 0.7. Sites with experimentally verified human-adaptive mutations and computationally identified markers are highlighted in red and green, respectively, in a, b; blue nodes indicate novel markers

Fig. 4. Correlation of root-to-tip divergence with evolution time.

a Correlation of representative H1N1 PB2 sequences from 1918 to 2009 with root-to-tip divergence. b Correlation of representative H3N2 PB2 sequences from 1968 to 2016 with root-to-tip divergence. The x axis represents the evolution time of the MRCA; the y axis represents the sequence divergence from the MRCA. Each dot corresponds to a PB2 protein sequence

Fig. 5. MCC tree and mutational path of seasonal H1N1 IAV.

a MCC tree of seasonal H1N1 IAVs circulated between 1918 and 2009. A total of 115 representative PB2 sequences were selected to reconstruct the tree by Bayesian phylogenetic inference. The evolutionary path from the MRCA to the most divergent descendant (A/California/6/2007) is highlighted in the tree. b Reconstructed mutational path from the MRCA to the most divergent descendant. The x and y axes represent the time scale and the mutations in the path, respectively. The median and 90% BCI of estimated date of each mutation are shown in the boxplot. Experimentally verified markers, computationally predicted markers, and novel markers are indicated in red, green, and blue, respectively

Fig. 6. MCC tree and mutational path of seasonal H3N2 IAV.

a MCC tree of seasonal H3N2 IAVs circulating between 1968 and 2016. A total of 170 representative PB2 sequences were selected to reconstruct the tree by Bayesian phylogenetic inference. The evolutionary path from the MRCA to the most divergent descendant (A/Hawaii/13/2016) is highlighted in the tree. b Reconstructed mutational path from the MRCA to the most divergent descendant. The x and y axes represent the time scale and the mutations in the path, respectively. The median and 90% BCI of estimated date of each mutation are shown in the boxplot. Experimentally verified markers, computationally predicted markers, and novel markers are indicated in red, green, and blue, respectively