Abstract
Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments. Through genetic analysis and functional characterization, we here show that the NS segment of H7N9 virus contains a unique G540A substitution, located within a previously undefined exonic splicing enhancer (ESE) motif present in the NEP mRNA of influenza A viruses. G540A supports virus replication in mammalian cells while retaining replication ability in avian cells. Host splicing regulator, SF2, interacts with this ESE to regulate splicing of NEP/NS1 mRNA and G540A substitution affects SF2–ESE interaction. The NS1 protein directly interacts with SF2 in the nucleus and modulates splicing of NS mRNAs during virus replication. We demonstrate that splicing of NEP/NS1 mRNA is regulated through a cis NEP-ESE motif and suggest a unique NEP-ESE may contribute to provide H7N9 virus with the ability to both circulate efficiently in avian hosts and replicate in mammalian cells.
Highlights
Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments
Unspliced M1 mRNA levels are downregulated when non-structural protein 1 (NS1) expression is decreased in A540G mutant virus infected cells (Fig. 3e). Mutation of this exonic splicing enhancer (ESE) did not affect NP mRNA levels during the early hours of virus infection (Fig. 3f). These results further demonstrate that the NEP ESE is associated with splicing of NS mRNAs, and that the G540A substitution in this ESE affects the ratio of NS1 to NEP mRNAs, leading to more NEP protein being expressed in the early hours of infection
We showed that full-length FLAG-tagged SF2 in nuclear extract (NE) prepared from plasmid transfected HEK293T cells is able to bind to a probe derived from nucleotides 529–548, which cover the NEP-ESE site; the specificity of the shifted complex is further verified by binding of anti-FLAG, but not the IgG antibody control, to tagged SF2 (Fig. 4b)
Summary
Influenza virus utilizes host splicing machinery to process viral mRNAs expressed from both M and NS segments. We demonstrate that splicing of NEP/NS1 mRNA is regulated through a cis NEP-ESE motif and suggest a unique NEP-ESE may contribute to provide H7N9 virus with the ability to both circulate efficiently in avian hosts and replicate in mammalian cells. NEP is required for coordination of viral ribonucleoprotein (RNP) nuclear export in the later stage of infection and it is suggested that virus adapts to utilize a poor 50-splice site to allow slow expression of NEP early in virus infection[20]. These studies indicate that regulation of the NS splicing process plays a key role in virus replication. It seems that splicing of NS mRNAs can be regulated by the NEP ESE and that mutation within this ESE may alter virus replication efficiency
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