Summary Many viruses shut off host gene expression to inhibit antiviral responses. Viral proteins and host proteins required for viral replication are typically spared in this process, but the mechanisms of target selectivity during host shutoff remain poorly understood. Using transcriptome-wide and targeted reporter experiments, we demonstrate that the influenza A virus endoribonuclease PA-X usurps RNA splicing to selectively target host RNAs for destruction. Proximity-labeling proteomics reveals that PA-X interacts with cellular RNA processing proteins, some of which are partially required for host shutoff. Thus, PA-X taps into host nuclear pre-mRNA processing mechanisms to destroy nascent mRNAs shortly after their synthesis. This mechanism sets PA-X apart from other viral host shutoff proteins that target actively translating mRNAs in the cytoplasm. Our study reveals a unique mechanism of host shutoff that helps us understand how influenza viruses suppress host gene expression.

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SUMMARYMany viruses globally shut off host gene expression to inhibit activation of cell-intrinsic antiviral responses. However, host shutoff is not indiscriminate, since viral proteins and host proteins required for viral replication are still synthesized during shutoff. The molecular determinants of target selectivity in host shutoff remain incompletely understood. Here, we report that the influenza A virus shutoff factor PA-X usurps RNA splicing to selectively target host RNAs for destruction. PA-X preferentially degrades spliced mRNAs, both transcriptome-wide and in reporter assays. Moreover, proximity-labeling proteomics revealed that PA-X interacts with cellular proteins involved in RNA splicing. The interaction with splicing contributes to target discrimination and is unique among viral host shutoff nucleases. This novel mechanism sheds light on the specificity of viral control of host gene expression and may provide opportunities for development of new host-targeted antivirals.

Tail-anchored (TA) proteins are a unique class of integral membrane proteins that possess a single C-terminal transmembrane domain and target post-translationally to the specific organelles at which they function. While significant advances have been made in recent years in elucidating the mechanisms and molecular targeting signals involved in the proper sorting of TA proteins, particularly to the endoplasmic reticulum and mitochondria, relatively little is known about the targeting of TA proteins to the plastid outer envelope. Here we show that several known or predicted plastid TA outer envelope proteins (OEPs) in Arabidopsis possess a C-terminal RK/ST sequence motif that serves as a conserved element of their plastid targeting signal. Evidence for this conclusion comes primarily from experiments with OEP7.2, which is a member of the Arabidopsis 7 kDa OEP family. We confirmed that OEP7.2 is localized to the plastid outer envelope and possesses a TA topology, and its C-terminal sequence (CTS), which includes the RK/ST motif, is essential for proper targeting to plastids. The CTS of OEP7.2 is functionally interchangeable with the CTSs of other TA OEPs that possess similar RK/ST motifs, but not with those that lack the motif. Further, a bioinformatics search based on a consensus sequence led to the identification of several new OEP TA proteins. Collectively, this study provides new insight into the mechanisms of TA protein sorting in plant cells, defines a new targeting signal element for a subset of TA OEPs and expands the number and repertoire of TA proteins at the plastid outer envelope.