Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein

Abstract

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.

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus

“…3A). Several studies have reported that the PA-X protein is involved in the inhibition of host protein expression (15,16,18,20,35,42,43). In order to generate a PA-X-deficient construct, the frameshift sequence was changed to two different sequences to potentially inhibit PA-X expression (UCC UUC AGA or AGC UUC AGA) (Fig.…”

“…In order to generate a PA-X-deficient construct, the frameshift sequence was changed to two different sequences to potentially inhibit PA-X expression (UCC UUC AGA or AGC UUC AGA) (Fig. 3A) (15,16,18,20,35,42,43). Importantly, none of the approaches used for the abolition of the ribosomal frameshift motif changed the PA amino acid sequence.…”

“…not able to detect PA-X expression by Western blotting, most likely due to the low frameshift efficiency (around 1.3%) (15). However, taking into account that the PA-X protein has a much stronger endonucleolytic activity than the PA protein (15,18) and that the amino acid sequence of the PA protein is the same in all the constructs, is very likely that the mutations introduced in the frameshift of the PA MUT construct, but not those introduced in the PA PMUT construct, decreased PA-X protein expression levels, leading to a lower inhibition of general gene expression mediated by the PA-X protein. One of the mechanisms used by the IAV NS1 protein to counteract innate immune responses is the binding to CPSF30, which leads to host gene expression suppression, including expression of IFN and ISGs with antiviral activities (28,30).…”

“…Altogether, these findings suggest that the PA-X protein interplay with the NS1 protein can slightly affect the viral polymerase activity, at least in the context of the pH1N1 LAIV virus backbone. However, the mechanism involving the reduction in the activity of the viral polymerase complex is not totally understood and could involve the interaction of the viral proteins with cellular host factors (18,44,45).…”

“…Since the influenza virus PA-X and NS1 proteins were identified, multiple functions for these viral factors have been described (14,15,18,29). Notably, the PA-X and NS1 from some IAV strains have the synergistic ability to block the host protein synthesis, although to do that, PA-X and NS1 use different mechanisms (14,15,17,18,29).…”