Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection

Abstract

The RNA modification N⁶-methyladenosine (m⁶A) modulates mRNA fate and thus affects many biological processes. We analyzed m⁶A across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m⁶A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m⁶A to RIOK3 promotes its translation, while loss of m⁶A in CIRBP promotes alternative splicing. Importantly, viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m⁶A in RIOK3 or CIRBP, respectively. Further, several transcripts with infection-altered m⁶A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m⁶A modification of host mRNAs to regulate infection.

Keywords: CIRBP; ER stress; Flaviviridae; RIOK3; RNA modification; epitranscriptome; innate immunity; m(6)A; signaling; viral infection.

Figure 1:. Flaviviridae infection alters m⁶A modification of specific transcripts.

(A) Schematic of the MeRIP-seq protocol used to identify differential m⁶A methylation following infection of Huh7 cells with DENV, ZIKV, WNV, and HCV. RNA was harvested at 48 hours post-infection (hpi) and experiments were performed in triplicate. (B) The number of peaks and genes with m⁶A peaks detected in ≥ 2 mock- or virus-infected samples (dark blue; MACS2 q-value < 0.05) and peaks that change during infection (light blue, |peak – gene Log₂FC| ≥ 1, adjusted p < 0.05). “Infection-annotated genes:” genes with known annotations for the Reactome Pathways ‘Infectious Disease’, ‘Unfolded Protein Response’, ‘Interferon Signaling’, or ‘Innate Immune Signaling’ in the database used by fgsea. “Infection-regulated genes:” genes that show a Log₂FC in gene expression ≥ 2 in RNA expression between mock- and virus- infected samples (adjusted p < 0.05). (C) The most significantly enriched motif in the MeRIP fractions across all samples (HOMER, p = 1e-831). (D) Metagene plot of “methylated” DRACH motifs (detected in a peak in at least two replicates) across transcripts in mock- and virus- infected cells. (E) The percent of genes with m⁶A peaks that changed expression with infection (|Log₂FC| ≥ 2, adjusted p < 0.05, N = 137) and genes that remained stable (|Log₂FC| < 0.5, adjusted p > 0.05, N = 7627) for transcripts with mean expression ≥ 50 reads. (F) (Left) MeRIP-RT-qPCR analysis of relative m⁶A level of transcripts with infection-altered m⁶A modification or controls (ACTB and MAVS) in DENV, ZIKV, and HCV-infected (48 hpi) Huh7 cells. (Right) RNA expression of these transcripts relative to GAPDH. Values in heatmap are the mean of 3 independent experiments. * p < 0.05, by unpaired Student’s t test. See also Figure S1 and Table S1 and S2.

Figure 2:. Flaviviridae infection alters m⁶A modification of RIOK3 and CIRBP mRNA through distinct cellular pathways.

(A and B) Coverage plot of MeRIP (color) and input (black) reads in (A) RIOK3 and (B) CIRBP transcripts in Huh7 cells infected with the indicated virus (48 hpi), as determined by MeRIP-seq. Representative of three biological replicates. Infection-altered m⁶A peaks are indicated in black under the transcript map. (C) (Left) MeRIP-RT-qPCR analysis of relative m⁶A level of RIOK3 and CIRBP in mock- and virus-infected (48 hpi) Huh7 cells. (Right) RNA expression of RIOK3 and CIRBP relative to HPRT1. (D) (Left) MeRIP-RT-qPCR analysis of relative m⁶A level of RIOK3 and CIRBP in mock- and HCV PAMP- transfected (8 h) Huh7 cells. (Right) RNA expression of RIOK3, CIRBP, as well as positive control transcripts IFNB1 and IFIT1 relative to HPRT1. (E) (Left) MeRIP-RT-qPCR analysis of relative m⁶A level of RIOK3 and CIRBP in mock- and thapsigargin-treated (TG; 16 h) Huh7 cells. (Right) RNA expression of RIOK3, CIRBP, and positive control transcripts HSPA5 and XBP1 relative to HPRT1. Values are the mean ± SEM of 6 (C-D), or 5 (E) biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired Student’s t test. n.s. = not significant. See also Figure S2 and Table S2 and S3.

Figure 3:. m⁶A promotes RIOK3 protein expression.

(A) (Left) Representative immunoblot of RIOK3 protein expression in mock- and virus-infected (48 hpi) Huh7 cells. (Right) Quantification of RIOK3 protein expression relative to tubulin. (B) Immunoprecipitation (IP) of RIOK3 from mock- and DENV-infected (48 hpi) Huh7 cells labeled with ³⁵S for 3 hours. IP fractions were analyzed by autoradiography (³⁵S) and immunoblotting. Representative of 3 biological replicates. (C) (Left) Representative immunoblot of RIOK3 protein expression in mock- and DENV-infected (48 hpi) Huh7 cells treated with non-targeting control (CTRL) or YTHDF1 siRNA. (Right) Quantification of RIOK3 protein expression relative to tubulin. (D) Schematic of WT and mutant m⁶A-null Renilla luciferase (RLuc) RIOK3 3’ UTR reporters that also express m⁶A-null Firefly luciferase (FLuc) from a separate promoter. RT-qPCR primers (F and R) are indicated with arrows. (E) MeRIP-RT-qPCR analysis of relative m⁶A level of stably expressed WT and m⁶A-mut RIOK3 3’ UTR reporter RNA in mock- and virus-infected (48 hpi) Huh7 cells. (F) Relative luciferase activity (RLuc/FLuc) in mock- and virus-infected (48 hpi) Huh7 cells stably expressing WT and m⁶A-mut RIOK3 3’ UTR reporters. Relative luciferase activity in uninfected cells was set as 1 for each reporter. Values are the mean ± SEM of 6 (A), 4 (C), 2 (E), or 5 (F) biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired Student’s t test. n.s. = not significant. See also Figure S3.

Figure 4:. m⁶A promotes alternative splicing of CIRBP.

(A) Schematic of CIRBP transcript isoforms with a focus on the alternatively spliced region (ASR). RT-qPCR primer locations are indicated with arrows (F_C-R_C: control CIRBP amplicon; F-R_L: long isoform specific; F-R_S: short isoform specific. (B) RT-qPCR analysis of short (S) and long (L) CIRBP RNA isoforms in mock- and virus-infected (48 hpi) Huh7 cells relative to control CIRBP amplicon. (C) RT-qPCR analysis of S and L CIRBP RNA isoforms in mock- and TG-treated (16 h) Huh7 cells. (D) (Left) Representative immunoblot of short (CIRBP-S) and long (CIRBP-L) CIRBP protein isoforms in mock- and virus-infected (48 hpi) Huh7 cells. (Right) Quantification of CIRBP protein isoform expression relative to tubulin. (E) (Left) Representative immunoblot analysis of CIRBP protein isoforms in mock- and TG-treated (500nM, 16 h) Huh7 cells. HSPA5 and GADD34 are positive controls. (Right) Quantification of CIRBP protein isoform expression relative to tubulin. (F) Schematic of WT and m⁶A-mut CIRBP splicing reporters. RT-qPCR primer locations (F_luc-R_luc: control; F-R_L: long isoform specific; F-R_s: short isoform specific) are indicated with arrows. (G) RT-qPCR analysis of CIRBP splicing reporter isoform expression (S and L) relative to control RLuc amplicon in Huh7 cells transfected with WT and m⁶A-mut constructs. Values are the mean ± SEM of 3 (B, D, E, G) or 5 (C) biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired Student’s t test. n.s. = not significant. See also Figure S4.

Figure 5:. RIOK3 and CIRBP regulate Flaviviridae infection.

(A) Focus-forming assay (FFA) of supernatants from DENV, ZIKV, or HCV-infected (72 hpi) Huh7 cells treated with non-targeting control (CTRL) or RIOK3 siRNA. (B) FFA of supernatants from DENV, ZIKV, or HCV-infected (72 hpi) Huh7 cells stably overexpressing FLAG-GFP or FLAG-RIOK3 (2 independent clones). (C) Immunoblot analysis of cell lines in (B). (D) FFA of supernatants harvested from DENV, ZIKV, or HCV-infected (72 hpi) Huh7 treated with CTRL or CIRBP siRNA. (E) FFA of supernatants from DENV, ZIKV, or HCV-infected (72 hpi) Huh7 cells stably overexpressing FLAG-GFP or the short (FLAG-CIRBP-S) or long (FLAG-CIRBP-L) isoforms of CIRBP (2 independent clones). (F) Immunoblot analysis of cell lines in (C). Values are the mean ± SEM of 4 (A and D), or 3 (B, E, G) biological replicates. Viral infections were performed at a multiplicity of infection of 0.2. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired Student’s t test. n.s. = not significant. See also Figure S5.

Figure 6:. Genes with infection-induced m⁶A alterations regulate Flaviviridae infection.

Heatmap of viral titers of supernatants harvested from DENV, ZIKV, or HCV-infected cells (48 hpi) treated with the indicated siRNAs. Data are presented as percentage of titer of each virus relative to cells treated with CTRL siRNA. Colors represent the mean of 3 biological replicates. Viral infections were performed at a multiplicity of infection of 0.2. * p < 0.05 by unpaired Student’s t test. See also Figure S6.