Influenza A virus is a human pathogen whose genome is comprised of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. Since nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

Three of the eight RNA segments encoded by the influenza A virus (IAV) undergo alternative splicing to generate distinct proteins. Previously, we found that host proteins hnRNP K and NS1-BP regulate IAV M segment splicing, but the mechanistic details were unknown. Here we show NS1-BP and hnRNP K bind M mRNA downstream of the M2 5′ splice site (5′ss). NS1-BP binds most proximal to the 5′ss, partially overlapping the U1 snRNP binding site, while hnRNP K binds further downstream and promotes U1 snRNP recruitment. Mutation of either or both the hnRNP K and NS1-BP-binding sites results in M segment mis-splicing and attenuated IAV replication. Additionally, we show that hnRNP K and NS1-BP regulate host splicing events and that viral infection causes mis-splicing of some of these transcripts. Therefore, our proposed mechanism of hnRNP K/NS1-BP mediated IAV M splicing provides potential targets of antiviral intervention and reveals novel host functions for these proteins.

Viral infection induces the expression of numerous host genes that impact the outcome of infection. Here we show that infection of human lung epithelial cells with Influenza A virus (IAV) also induces a broad program of alternative splicing of host genes. While these splicing-regulated genes are not enriched for canonical regulators of viral infection, we find that many of these genes do impact replication of IAV. Moreover, in several cases, specific inhibition of the IAV-induced splicing pattern also attenuates viral infection. We further show that approximately a quarter of the IAV-induced splicing events are regulated by hnRNP K, a host protein required for efficient splicing of the IAV M transcript in nuclear speckles. Finally, we find an increase in hnRNP K in nuclear speckles upon IAV infection, which may alter accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.

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