Genomic Signatures for Avian H7N9 Viruses Adapting to Humans

Abstract

An avian influenza A H7N9 virus emerged in March 2013 and caused a remarkable number of human fatalities. Genome variability in these viruses may provide insights into host adaptability. We scanned over 140 genomes of the H7N9 viruses isolated from humans and identified 104 positions that exhibited seven or more amino acid substitutions. Approximately half of these substitutions were identified in the influenza ribonucleoprotein (RNP) complex. Although PB2 627K of the avian virus promotes replication in humans, 45 of the 147 investigated PB2 sequences retained the E signature at this position, which is an avian characteristic. We discovered 10 PB2 substitutions that covaried with K627E. An RNP activity assay showed that Q591K, D701N, and M535L restored the polymerase activity in human cells when 627K transformed to an avian-like E. Genomic analysis of the human-isolated avian influenza virus is crucial in assessing genome variability, because relationships between position-specific variations can be observed and explored. In this study, we observed alternative positions that can potentially compensate for PB2 627K, a well-known marker for cross-species infection. An RNP assay suggested Q591K, D701N, and M535L as potential markers for an H7N9 virus capable of infecting humans.

Fig 1. Web logos for H7N9 genome displaying at least 7 substitutions.

Positions accompanied by red dots are among the 47 species-associated signature positions listed in Table 2. The leading three logos for PA-X and PA are identical, as the two proteins share the 191 amino acids at the N terminal. Sequence counts used in each of these logos are included in the parentheses as PB2 (147), PB1 (137), PB1-F2 (134), PA (139), PA-X (132), HA (142), NP (140), NA (141), M1 (143), M2 (142), NS1 (141), NS2 (142). The Y-axis shows the residue frequency for each residue, which adds up to 1.0.

Fig 2. Stereography of influenza A H7N9 PB2 residue-varying positions listed in Table 3.

(A and B) Simulated H7N9 PB2 stereography for A/Taiwan/4-CGMH2/2014(H7N9) viral PB2 is shown in the ribbon mode. (C and D) The distribution of electrostatic potential on the protein surface (blue = relative positive charge; red = relative negative charge). Protein modeling was performed using SWISS-MODEL by applying Coulomb’s law in Chimera according to the full-length PB2 of A/little yellow-shouldered bat/Guatemala/060/2010(H17N10) (PDB ID 4WSB).

Fig 3. Statistics for the RNP activity assay of the influenza A H7N9 PB2.

(A) RNP activity for the H7N9 virus in 293T cells. The RNP complexes exhibiting combinations of the avian signature PB2 627E and its covarying residues (positions labeled with asterisks in Table 3) from the A/Anhui/1/2013(H7N9) virus were cotransfected into the 293T cells. CAT ELISA was performed 48 h after transfection to detect protein expression levels of the virus-like CAT reporter, thereby signaling the viral RNP activity. The error bars show the SEM from 3 independent experiments. Two-way ANOVA and the GraphPad Prism Software were used to statistically support the observed disparity. (B) Each component of the viral RNP complex and NP was detected using Western blotting.