Permissive secondary mutations enable the evolution of influenza oseltamivir resistance

Abstract

The His274-->Tyr274 (H274Y) mutation confers oseltamivir resistance on N1 influenza neuraminidase but had long been thought to compromise viral fitness. However, beginning in 2007-2008, viruses containing H274Y rapidly became predominant among human seasonal H1N1 isolates. We show that H274Y decreases the amount of neuraminidase that reaches the cell surface and that this defect can be counteracted by secondary mutations that also restore viral fitness. Two such mutations occurred in seasonal H1N1 shortly before the widespread appearance of H274Y. The evolution of oseltamivir resistance was therefore enabled by "permissive" mutations that allowed the virus to tolerate subsequent occurrences of H274Y. An understanding of this process may provide a basis for predicting the evolution of oseltamivir resistance in other influenza strains.

Fig. 1

H274Y decreases NA surface expression and viral fitness for H1N1 lab strains. (A) Plasmids encoding wild-type (WT, black squares) or H274Y (gray circles) NAs were transfected into 293T cells. Cells were assayed for total surface NA activity in a nonlysing buffer. (B) The same cells were assayed by flow cytometry for mean surface NA expression using a monoclonal antibody against the PR8 tetramer. (C) Surface activity of WT, H274Y, or H274Y-R194G WSN NA at 18 hours. All four graphs show mean ± SEM for at least three independent transfections. (D) An estimated 50 infectious particles of each color of WSN-PB1flank-eGFP and/or mCherry carrying the indicated NA mutations were infected into 2.5 × 10⁵ A549-CMV-PB1 cells. After 46 to 50 hours, the cells were analyzed by flow cytometry to quantify virus growth. Numbers give percentages of cells in each gate; one representative plot is shown for each virus pair. (E) Relative fitness (ratio of Malthusian growth parameters) with respect to WT calculated from at least eight replicates. Shown are geometric means and 95% confidence intervals.

Fig. 2

Total surface-expressed activity for WT (black squares) and H274Y (gray circles) NAs from (A) three seasonal human H1N1 strains and (B) the pandemic swine-origin A/California/4/2009 (H1N1) strain. All graphs show mean ± SEM for at least three replicates.

Fig. 3

Identification of mutations that buffer defects in NA surface expression and viral fitness caused by H274Y in seasonal human H1N1. (A) Maximum-likelihood phylogenetic tree showing NC99 and all seasonal H1N1 strains from 2006 or later. Based on a maximum-likelihood reconstruction, branches with H274Y are colored red, whereas those without are colored black. Indicated are the first five mutations on the path from NC99 to the large group of H274Y viruses. Bootstrap percentages are shown for key nodes. Branches with V234M are shaded dark blue, and those with both V234M and R222Q are shaded light blue. One of the isolated H274Y sequences contained an independent appearance of V234M as indicated. The NC99 and SI06 sequences are labeled. (B) Surface-expressed activity at 20 hours for NC99 NAs with sequential addition of the five reconstructed mutations, both with and without H274Y. (C) Surface-expressed activity and mean staining intensity at 20 hours for NC99 NAs containing a C-terminal epitope tag. (D) An estimated 50 infectious particles of PB1flank-eGFP with the indicated NC99 NAs and the remaining genes from TX91 were infected into 2.5 × 10⁵ MDCK-SIAT1-CMV-PB1 cells, and the viral titer in the supernatent was determined at the indicated times. All graphs show mean ± SEM for at least three replicates.