Date Published: February 5, 2016
Publisher: Public Library of Science
Author(s): Denys A. Khaperskyy, Summer Schmaling, Jonah Larkins-Ford, Craig McCormick, Marta M. Gaglia, Peter Palese.
Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus.
Inhibition of host gene expression, termed “host shutoff”, is thought to enable viruses to simultaneously inhibit innate immune responses and provide preferential access for viral mRNAs to the cellular translation machinery. Influenza A virus (IAV) has long been known to carry out host shutoff, and multiple shutoff mechanisms have been reported for this virus, including translation blockade , inhibition of polyadenylation and nuclear export of host pre-mRNAs by the IAV NS1 protein , and degradation of the host RNA polymerase II complex . Because some of these mechanisms are specific to certain IAV strains , it has long been suspected that more universal IAV host shutoff mechanisms exist. The recent discovery of the highly conserved RNA endonuclease PA-X  has prompted re-examination of established models of IAV host shutoff. Viruses from several divergent families use virus-encoded RNA endonucleases to broadly degrade host mRNAs and reduce host protein production [5–9]. Although host shutoff ribonucleases (RNases) generally have broad specificity in vitro, several studies have shown unexpected selectivity for different types of host transcripts [10–15]. PA-X limits accumulation of host mRNAs and proteins in infected cells and suppresses host responses to infection [5,16–19], but the mechanistic determinants of selectivity, cleavage and degradation are not yet known.
Our study provides new mechanistic insights into the specificity of PA-X, the most recently identified viral host shutoff nuclease. We demonstrate here for the first time that PA-X is recruited to the nucleus, selectively targets a subset of host transcripts, and is not active against viral mRNAs. Moreover, we uncover a novel route of target discrimination by a viral RNase, which takes advantage of the divergent mRNA biogenesis mechanisms that generate viral and host transcripts. We demonstrate that PA-X shares some specificity features with other host shutoff nucleases, as it selectively targets products of cellular RNA Pol II, while sparing Pol I and Pol III transcripts, and requires host RNases to complete RNA degradation. Interestingly, the mechanism for PA-X targeting of Pol II transcripts is not related to the translatability of the mRNAs. Instead, PA-X targeting is directly linked to synthesis by RNA Pol II complex or early processing events unique to Pol II transcripts. Moreover, we find that PA-X likely degrades RNAs in the nucleus, because it accumulates in this compartment and affects both the nuclear and cytoplasmic fraction of its target RNAs. Both the host shutoff activity of PA-X and the nuclear localization function of the C-terminal X-ORF are dependent on the presence of a set of basic residues in the X-ORF, indicating a correlation between nuclear localization and RNA targeting. Thus, the mechanism of PA-X targeting may be different from other shutoff RNases because it is tightly linked to the mechanism of biogenesis of host and viral mRNAs in the nucleus.