Many viruses block host gene expression to take over the infected cell. This process, termed host shutoff, is thought to promote viral replication by preventing antiviral responses and redirecting cellular resources to viral processes. Several viruses from divergent families accomplish host shutoff through RNA degradation by endoribonucleases. However, viruses also need to ensure expression of their own genes. The influenza A virus endoribonuclease PA-X solves this problem by sparing viral mRNAs and some host RNAs necessary for viral replication. To understand how PA-X distinguishes between RNAs, we characterized PA-X cut sites transcriptome-wide using 5′ rapid amplification of complementary DNA ends coupled to high-throughput sequencing. This analysis, along with RNA structure predictions and validation experiments using reporters, shows that PA-Xs from multiple influenza strains preferentially cleave RNAs at GCUG tetramers in hairpin loops. Importantly, GCUG tetramers are enriched in the human but not the influenza transcriptome. Moreover, optimal PA-X cut sites inserted in the influenza A virus genome are quickly selected against during viral replication in cells. This finding suggests that PA-X evolved these cleavage characteristics to preferentially target host over viral mRNAs in a manner reminiscent of cellular self versus non-self discrimination.
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All primary data (including uncropped gel and blot images) except for 5′ RACE-seq data are publicly available as a Figshare collection (https://doi.org/10.6084/m9.figshare.c.6616663)68. The raw 5′ RACE-seq sequences and identified cut sites are deposited in the NCBI Gene Expression Omnibus database (GSE207253).
All Python scripts used, including the PyDegradome 2.0 code, can be found in our laboratory’s GitHub page, https://github.com/mgaglia81/PyDegradome (ref. 69). The workflow explaining the use of the PyDegradome 2.0 code can be found in the Methods section (‘PyDegradome and other Python analyses’). The method of Reed and Muench44 was used to calculate virus titres using the Bloom Laboratory Python script at https://github.com/jbloomlab/reedmuenchcalculator (ref. 45). The RNA structure prediction software LinearFold52 is now available at https://linearfold.eecs.oregonstate.edu/. WebLogo56 is made available by the Computational Genomics Research Group (University of California, Berkeley) at https://weblogo.berkeley.edu/logo.cgi.
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We thank A. Tai and the personnel of the Tufts University Core Facility–Genomics Core for help with the sequencing; C. McCormick and D. Khaperskyy (Dalhousie University, Halifax, NS, Canada) and members of their laboratories for their advice and input;. C. McCormick (Dalhousie University, Halifax, NS, Canada), B. Glaunsinger (University of California, Berkeley, CA, USA), R. Webby (St Jude’s Children Research Hospital, Memphis, TN, USA), G. Dreyfuss (University of Pennsylvania, Philadelphia, PA, USA), J. Bloom (Fred Hutchinson Cancer Center, Seattle, WA, USA) and S. Lakdawala (Emory University School of Medicine, Atlanta, GA, USA) for constructs; M. G. Blanco (University of Virginia, Charlottesville, VA, USA) for providing the northern blot protocol; B. Moss (National Institute of Health, Bethesda, MD, USA) for cell lines; and J. Coffin, C. Moore, K. Munger (Tufts University, Boston, MA, USA) and members of the Gaglia Laboratory for critical reading of the manuscript. This work was supported by NIH grant R01 AI137358 (to M.M.G.). L.G. was supported by NIH F31 AI154587 and T32 GM007310. C.H.R. was partially supported by the Applied Mathematics Program of the US DOE Office of Science Advanced Scientific Computing Research under contract number DE-AC02-05CH11231. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
The authors declare no competing interests.
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Extended Data Fig. 1 Characteristics of the system used to identify PA-X cut sites transcriptome-wide.
(a) Strategy used to engineer a virus that lacks PA-X, PR8-PA(∆X), compared to WT PR8. (b) Protein lysates of XRN1 knock out A549 cells infected with WT PR8 or PR8-PA(∆X), or mock infected, were probed with antibodies against PR8 PA or β-tubulin as a loading control. Images are representative of 2 experiments. (c) Protein lysates of WT or XRN1 knock out (ko) A549 cells were probed with antibodies against Xrn1 or β-tubulin as a loading control to check for loss of XRN1. Images are representative of 2 experiments. (d, e) Representation of individual chromosomal positions in the 5′ RACE-seq data. For each sample, reads with their 5′ end mapping to the same nucleotide were counted and plotted to compare different datasets: (d) mock samples from replicate 1 vs. replicate 2, (e) replicate 1 WT PR8 vs. PR8-PA(∆X) (top), WT PR8 vs. mock infected (middle) and PR8-PA(∆X) vs. mock infected (bottom). For each plot, light blue dots correspond to locations that are unique to one sample, while red and black dots correspond to locations that are common in the two samples. Red dots represent locations that have two-fold or more reads in the sample on the x axis vs. than in the sample on the y axis. Similar plots were obtained when comparing other replicates. (f) PyDegradome was run on concatenated read files comparing WT PR8 vs. PR8-PA(∆X) infections using different parameters as indicated on the x axis (see Methods for parameter explanation). cl = confidence levels, mf = multiplicative factor. The window of analysis was 4 nucleotides in all cases. The total number of cut sites identified by PyDegradome (red circles, left y axis) and the percentage of shared cut sites also found as concatenated cut sites (blue diamonds, right y axis) were plotted for each set of parameters. Panel a adapted with permission from 4, Elsevier.
(a, b) XRN1 knock out A549 cells were infected with WT PR8 or PR8-PA(∆X), or mock infected. (a) RNA was isolated, 5′ RACE was performed using primers specific for BCAP31, TUBA1B, INSIG1 or SLC7A5, and the PCR products were run on an agarose gel. Primers were positioned ~ 200–300 nucleotides downstream of the predicted cut sites. The predicted size of DNA bands coming from cut sites identified by PyDegradome are indicated by red dotted arrows. (b) PR8 HA RNA levels were quantified by qRT-PCR, normalized to 18S and plotted as mean ± standard deviation. AU, arbitrary units; n.d. not defined; ns, not significant, One-way ANOVA with Dunnett’s multiple comparison test. n = 3 independent experiments. (c) HEK293T ishXRN1 cells were treated with no drug or doxycycline for 3-4 days to induce shRNAs against XRN1, then protein lysates were collected and probed with antibodies against XRN1, or β-tubulin as a loading control to check for efficient XRN1 knock down. Images are representative of 3 experiments. (d, e) HEK293T ishXRN1 cells were treated with doxycycline for 3-4 days to induce knock down of XRN1, then transfected with luciferase reporters containing 99 bp insertions from the indicated genes, and where indicated, with PR8 PA-X. (d) RNA was extracted and luciferase mRNA levels were quantified by qRT-PCR, normalized to 18S and plotted as mean ± standard deviation. AU, arbitrary units. luc + INSIG1/SLC7A5 n = 2, luc + STOML2/YKT6/TUBA1B n = 3, luc + BCAP31 n = 4, luc + repeat n = 8, luc n = 9 independent experiments. (e) The RNA was also used to run 5′ RACE. Expected sizes of DNA bands coming from cut sites in the introduced target sequences are indicated by red arrows, while the blue arrow in D indicates the size of the original luciferase cut site fragment. For all gels, the DNA bands were purified and sequenced to confirm their identities. Images are representative of 3 experiments or 2 experiments for luciferase + INSIG1/SLC7A5. The SLC7A5 sequence was only consistently cut when inside the luciferase reporter.
Extended Data Fig. 3 The cut sites identified by PyDegradome are specific to PA-X and conserved across multiple influenza strains.
(a–c) HEK293T ishXRN1 cells were treated with doxycycline for 3-4 days to induce knock down of XRN1, then transfected with luciferase reporters containing 99 bp insertions from the indicated genes, and where indicated, with WT PA-X from the PR8 strain (a–c), the catalytic mutant PR8 PA-X D108A (a), PR8 PA with a mutation to reduce frameshifting and prevent PA-X production (PA(fs)) (a), herpes simplex virus 1 (HSV-1) vhs or Kaposi’s sarcoma-associated herpes virus (KSHV) SOX (b), or WT PA-X from the Perth influenza strain (c). RNA was extracted and used to run 5′ RACE. Expected sizes of DNA bands coming from cut sites in the introduced target sequences are indicated by the red dotted arrows. (d) XRN1 knock out A549 cells were infected with WT PR8, Perth or pH1N1pdm09 or the corresponding PA(∆X) mutants, or mock infected. 5′ RACE was then performed using primers specific for STOML2 or YKT6 ~ 250–300 nt downstream of the predicted cut sites. The PCR products were run on an agarose gel. The predicted size of DNA bands coming from cut sites identified by PyDegradome are indicated by the red dotted arrows. For all gels, the DNA bands were purified and sequenced to confirm their identities, and images are representative of 3 experiments. (e) Protein alignment of PA-X from the three different influenza strains PR8, H1N1pdm09 and Perth, generated using Clustal Omega69.
Extended Data Fig. 4 PA-X preferentially cleaves RNA at GCUG tetramers based on the concatenated cut site analysis.
(a, b) WebLogo56 representation of base enrichment around PA-X cut sites (WT PR8 vs. PR8-PA(∆X), a) or around control sites enriched in the PR8-PA(∆X) sample (PR8-PA(∆X) vs. WT PR8, b) for sites predicted by PyDegradome using the concatenated approach. (c) Percentage of PA-X cut sites containing GCUG or a tetramer with one nucleotide difference from GCUG, for sites identified by PyDegradome using the concatenated approach. // indicates the location of the cut, that is GCU//G indicates that PA-X cuts between the U and the G. (d) Further breakdown of the PA-X cut sites containing a tetramer with one nucleotide difference from GCUG around the cut site (marked by an x). (e, f) HEK293T ishXRN1 cells were treated with doxycycline for 3-4 days to induce knock down of XRN1, then transfected with luciferase reporters containing 99 bp insertions from the indicated genes, with or without PR8 PA-X. (e) RNA was extracted and luciferase mRNA levels were quantified by qRT-PCR, normalized to 18S and plotted as mean ± standard deviation. Left plot shows mRNA levels of each reporter in the absence of PA-X. Right plot shows mRNA levels of each reporter in the presence vs. absence of PA-X. AU, arbitrary units. n = 3 independent experiments. (f) The RNA was also used to run 5′ RACE. Expected sizes of DNA bands coming from cut sites in the introduced target sequences are indicated by the red dotted arrows. For both gels, DNA bands were purified and sequenced to confirm their identities, and images are representative of 3 experiments.
Extended Data Fig. 5 PA-X preferentially cleaves RNA within a hairpin loop structure in transfected and infected cells.
(a) HEK293T ishXRN1 cells were treated with doxycycline for 3-4 days to induce knock down of XRN1, then transfected with luciferase reporters containing insertions of the indicated lengths from the BCAP31 and SLC7A5 genes, with or without PR8 PA-X. RNA was extracted and used to run 5′ RACE. Expected sizes of DNA bands coming from cut sites in the introduced target sequences are indicated by the red dotted arrows (~250 bp for 99 bp constructs, ~ 220 bp for 51 bp constructs, ~ 210 bp for 27 bp constructs and ~ 205 bp for the 15 bp constructs). (b) HEK293T ishXRN1 cells were treated with doxycycline for 3-4 days to induce knock down of XRN1, then transfected with luciferase reporters containing 15 bp insertions from the STOML2 gene with or without the indicated mutations. 24 hours post transfection, cells were infected with WT PR8 or PR8-PA(∆X), or mock infected overnight. RNA was then extracted and used to run 5′ RACE. Expected sizes of DNA bands coming from cut sites in the introduced target sequences are indicated by the red dotted arrow, while the blue arrow indicates the size of the original luciferase cut site fragment. For all gels, the DNA bands were purified and sequenced to confirm their identities, and images are representative of 3 experiments (b) or 2 experiments (a).
(a–c) Percentage of PA-X cut sites found within introns or exons for sites identified by PyDegradome using the shared cut sites approach (a and c, left) or the concatenated approach (A and C, right), compared to the percentage of reads found in exons vs. introns (b). The reads used in B are from WT PR8 replicate 1 as an example, but other samples have similar read distribution. The difference between reads and PA-X cut site distribution in introns vs. exons is statistically significant (P < 0.001), but the difference between reads and ∆X specific sites distribution in the concatenated analysis is not (P > 0.2), Chi-Square Test for Goodness of Fit with degree of freedom 1. While only 3% of PR8-PA(∆X) fragments from the shared analysis mapped to introns (0.025 < P < 0.05 compared to reads distribution, Chi-Square Test for Goodness of Fit with degree of freedom 1), this may stem from the very low number of sites, 37, found with this method. (d–f) XRN1 knock out A549 cells were infected with WT PR8 or PR8-PA(∆X), or mock infected. 5′RACE was performed and PCR products were run on an agarose gel. The boxes show diagrams of the BCAP31, STOML2 or SLC7A5 genes and the positions of the reverse primers for 5′ RACE. Top gels are the same gels as Fig. 2b and Extended Data Fig. 3a, and are included for comparison. Red dotted arrows indicate the size of fragments originating from previously validated cut sites. Bottom gels show products obtained using reverse primers in the intron (light purple primers), and red dotted arrows indicate the predicted size of PCR products that would appear if PA-X cleaved unspliced pre-mRNAs. White arrowhead indicates fragments that map to exon/intron junctions. New gel images are representative of 3 experiments.
Extended Data Fig. 7 PA-X only cleaves STOML2 and YKT6 preferred sequences if they are located within exons.
(a) Diagram of the luciferase reporters tested. Green and magenta arrows indicate positions of 5′ RACE PCR reverse primers, vertical arrows indicate location of predicted cut sites. (b–d) Testing of luciferase reporters with sequence insertions in the introns, as shown in diagram in A. HEK293T ishXRN1 cells were treated with doxycycline for 3-4 days to induce knock down of XRN1, then transfected with luciferase reporters containing the 99 bp insertions in the indicated genes either in an exon or an intron, with or without PR8 PA-X. (b) RNA was extracted and luciferase mRNA levels were quantified by qRT-PCR, normalized to 18S and plotted as mean ± standard deviation. AU, arbitrary units; n = 3 except for luc + STOML2/YKT6 in exon n = 2, each independent experiments. The top graph shows levels of the processed mRNA (obtained using two primers that bind in exons). The bottom graph shows levels of the unspliced pre-mRNA (obtained using one primer in an exon and one primer in the intron). (c, d) RNA was also used to run 5′ RACE. PCR products were separated on an agarose gel. The top gel in C and the gel in D represent products obtained using the green arrow primer from A, and the bottom gel in C using the magenta arrow primer from A. Blue dotted arrow indicates size of PCR products originating from the original luciferase cut site, red dotted arrow indicates the predicted sizes of PCR products that would originate from the sequence inserted in the introns. Gel images are representative of 3 experiments.
The percentage of tetramers in the human and viral genomes and transcriptomes was calculated by counting the number of each tetramer and dividing it by the total number of tetramers in the sequence (that is length of the sequence minus 3). Percentages for each tetramer are plotted to visualize which tetramers are more abundant in the human genome vs. transcriptome (a) and the human transcriptome vs. the transcriptome of 3 influenza A virus strains (c-e). Each dot represents a specific tetramer, with the red dot representing the GCUG tetramer. (b) The percentage of GCUG tetramers is plotted for each influenza mRNA (that is the positive strand, purple), and for each influenza genomic RNA (that is negative strand, blue). Data are plotted as mean ± standard deviation, with each symbol representing a different influenza strain.
(a, b) XRN1 knock out A549 cells were infected with WT PR8 or PR8-PA(∆X) (a), or WT H1N1pdm09 or H1N1pdm09-PA(∆X) (b), or mock infected. RNA was extracted to run 5′ RACE using primers ~ 150–200 nt downstream of GCUG sites that are predicted to be in a hairpin loop in the indicated viral mRNAs. Red dotted arrows indicate the predicted sizes of PCR products that would originate from cleavage at these GCUG sites. Gel images are representative of 3 experiments. (c) MDCK cells were infected at MOI 0.05 with the indicated viruses (see Fig. 5f for details on virus construction). Supernatants were collected at 1, 8, 24 and 48 hours post infection and viral titers were quantified by TCID50. n = 2 independent experiments.
(a) PyDegradome was run for each replicate on WT PR8 vs. PR8-PA(∆X) infection samples using different parameters as indicated on the x axis (see Methods). cl = confidence levels, mf = multiplicative factor. The window of analysis was 4 nucleotides in all cases. The percentage of cut sites shared between all three replicates was plotted for each set of parameters. (b) Histogram of the number of reads at the cut site for PA-X sites (PyDegradome comparison: WT PR8 vs. PR8-PA(∆X)) found in one (light grey), two (dark grey), or three replicates (‘all’, magenta), or through the concatenated analysis (‘concat’, orange). (c) Number of PA-X cut sites (orange squares) or RNA fragments enriched in the PR8-PA(∆X) infected cells (purple circles) identified by PyDegradome relative to the number of input reads. (d) Percentage of sites identified by PyDegradome in at least one replicate that are located within the first 20 nucleotides of a gene when comparing WT PR8 vs.PR8-PA(∆X), PR8-PA(∆X) vs. WT PR8, WT PR8 vs. mock and PR8-PA(∆X) vs. mock.
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Gaucherand, L., Iyer, A., Gilabert, I. et al. Cut site preference allows influenza A virus PA-X to discriminate between host and viral mRNAs. Nat Microbiol 8, 1304–1317 (2023). https://doi.org/10.1038/s41564-023-01409-8
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