Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs

Gaucherand, Léa; Iyer, Amrita; Gilabert, Isabel; Rycroft, Chris H.; Gaglia, Marta Maria

doi:10.1038/s41564-023-01409-8

Cited by 6 publications

(12 citation statements)

References 69 publications

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“…In the past, this possibility has not been heavily investigated, since all studies have indicated that PA-X is active upon ectopic expression in uninfected cells, in the absence of other viral proteins. Also, our previous analyses also suggest that it has similar RNA targets in infected and uninfected cells(4–6). To test the hypothesis that other influenza proteins potentiate PA-X activity, we compared mRNA downregulation by ectopically expressed WT PA-X alone or by the NatA-modified PA-X E2A mutants expressed together with PR8 PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, or NEP (Fig.…”

Section: Resultsmentioning

confidence: 55%

“…Thus, it is possible that PA-X targets are retained in the nucleus by NS1, allowing for an increase in mRNA degradation by PA-X. In addition, we have also shown that PA-X preferentially cleaves GCUG tetramers located in single-stranded regions of hairpin loops(6). As NS1 binds double-stranded RNA(57,58), it could stabilize the hairpin region of PA-X targets, allowing for optimal substrate targeting by PA-X.…”

Section: Discussionmentioning

confidence: 91%

“…The E2 mutations in the pCR3.1-PA-X-eGFP constructs with PA-Xs from PR8, Perth, and H1N1pdm09 were generated using the QuikChange II site-directed mutagenesis kit (Agilent). The PR8 pHW-PA(ΔX) plasmid was generated from pHW193 by introducing mutations that reduce frameshifting events and add a premature stop codon in the PA-X reading frame, but that are silent in the PA reading frame, as previously described(5,6).…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type influenza A virus A/Puerto Rico/9/1934 H1N1 (PR8) and the mutant recombinant virus PR8 PA(∆X) were generated using the 8-plasmid reverse genetic system(62) as previously described (4)(5)(6). Viral stocks were produced in MDCK cells and infectious titers were determined by plaque assays in MDCK cells using 1.2% Avicel (FMC BioPolymer) overlays (63).…”

Section: Viruses and Infectionsmentioning

confidence: 99%

“…the global reduction of gene expression during infection (1,3,4). PA-X is an endoribonuclease (endoRNase) (1,4,5), and it carries out host shutoff by degrading host mRNAs, while largely sparing viral RNAs (4,6). While previous studies by us and others have dissected the specificity of RNA targeting by PA-X, we still have limited information on how PA-X functions.…”

Section: Introductionmentioning

confidence: 99%

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N-terminal acetylation separately promotes nuclear localization and host shutoff activity of the influenza A virus ribonuclease PA-X

Daly,

Myasnikov,

Gaglia

2023

Preprint

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To counteract host antiviral responses, influenza A virus triggers a global reduction of cellular gene expression, a process termed “host shutoff.” A key effector of influenza A virus host shutoff is the viral endoribonuclease PA-X, which degrades host mRNAs. While many of the molecular determinants of PA-X activity remain unknown, a previous study identified a requirement for N-terminal acetylation of PA-X for its host shutoff activity, but did not address how this modification promotes activity. Here, we report that PA-X N-terminal acetylation has two functions that can be separated based on the position of the acetylation, on the first amino acid, i.e. the initiator methionine, vs. the second amino acid following initiator methionine excision. Modification at either site is sufficient to ensure correct PA-X localization to the nucleus. However, modification at the second amino acid is not sufficient for host shutoff activity of ectopically expressed PA-X, which requires N-terminal acetylation of the initiator methionine specifically. Interestingly, during infection, N-terminal acetylation of PA-X at any position results in PA-X host shutoff activity, and this is, in part, due to a functional interaction with the influenza protein NS1. This reveals an unexpected role for other viral proteins in PA-X activity. Our studies highlight the multifaceted nature of PA-X N-terminal acetylation in its ability to regulate the host shutoff activity of PA-X through multiple avenues.

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Section: Resultsmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Viruses and Infectionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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N-terminal acetylation separately promotes nuclear localization and host shutoff activity of the influenza A virus ribonuclease PA-X

Daly,

Myasnikov,

Gaglia

2023

Preprint

Self Cite

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How influenza shuts down host transcription

Rivera-Cardona

Brooke

2023

Nat Microbiol

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Recruitment of multi-segment genomic RNAs by Bluetongue virus requires a preformed RNA network

Sung,

Phelan,

Luo

et al. 2024

Nucleic Acids Research

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How do segmented RNA viruses correctly recruit their genome has yet to be clarified. Bluetongue virus is a double-stranded RNA virus with 10 segments of different sizes, but it assembles its genome in single-stranded form through a series of specific RNA–RNA interactions prior to packaging. In this study, we determined the structure of each BTV transcript, individually and in different combinations, using 2′-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP identified RNA structural changes during complex formation and putative RNA–RNA interaction sites. Our data also revealed a core RNA-complex of smaller segments which serves as the foundation (‘anchor’) for the assembly of a complete network composed of ten ssRNA segments. The same order of core RNA complex formation was identified in cells transfected with viral RNAs. No viral protein was required for these assembly reactions. Further, substitution mutations in the interacting bases within the core assemblies, altered subsequent segment addition and affected virus replication. These data identify a wholly RNA driven reaction that may offer novel opportunities for designed attenuation or antiviral therapeutics.

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Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs

Cited by 6 publications

References 69 publications

N-terminal acetylation separately promotes nuclear localization and host shutoff activity of the influenza A virus ribonuclease PA-X

N-terminal acetylation separately promotes nuclear localization and host shutoff activity of the influenza A virus ribonuclease PA-X

How influenza shuts down host transcription

Recruitment of multi-segment genomic RNAs by Bluetongue virus requires a preformed RNA network

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