Abstract
Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3′ splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.
Highlights
Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans
While the avian virus is not adapted to efficient growth in cultured human cells and causes a non-permissive infection, the seasonal human virus replicates efficiently
We performed proteome-wide comparative pulse-labeling experiments by combining labeling with AHA and SILAC (Fig. 1a): cells incorporate AHA instead of methionine into newly synthesized proteins when the cell culture medium is supplemented with this bioorthogonal amino acid
Summary
Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. We combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a humanand a bird-adapted IAV strain and observe striking differences in viral protein synthesis. In the human upper respiratory airway epithelium, the dominant linkage is of α2,6 type, to which human-adapted HA binds Despite these differences in receptor binding, many avian viruses are internalized by human cells and initiate expression of the viral genome. Such infections typically lead to an abortive, nonproductive outcome in mammalian cell lines. Studies used radioactive pulse labeling to monitor protein synthesis in IAV-infected cells[28,29]. In combination with SILAC, AHA labeling can be used to quantify proteome dynamics with high temporal resolution[34,35,36]
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