Fig 1.
HSV-1 infection induces widespread and dynamic APA changes.
(A) A scatter plot of HSV-1-induced significant APA changes in HeLa cells (FDR < 0.05, at least 15% of the transcripts shifted). HSV0.p or HSV12.p: read counts for the proximal PAS in cells at 0 or 12 hpi; HSV0.d or HSV12.d: read counts for the distal PAS in cells at 0 or 12 hours post infection. DtoP: a distal to proximal shift; PtoD: a proximal to distal shift. Intron: shifts involving an intronic PAS. UTR: both PAS are located in the 3’ UTR. (B) Relative read count changes at proximal (.p) or distal (.d) PAS in the four groups of APA shifts. (C) A heat map showing the APA index (proximal/distal read count ratio) of all the genes with significant APA shifts as shown in (A). Data was scaled by row. Color bars on the left denote the 6 groups that displayed similar kinetic patterns. (D-F) PAS-seq tracks of example genes.
Fig 2.
HSV-1-induced APA changes occur, at least in part, co-transcriptionally.
(A) 4sU-seq signals of genes that displayed DtoP APA shifts in HSV-1-infected HeLa cells (8 hpi). TSS: transcription start site. TES: transcription end site. P values for the difference between the 4sU-seq signals at each position in mock and HSV1-infected cells were calculated using Wilcoxon rank sum test and plotted as a color-coded bar below the plot. The color scheme is shown in the inset. (B) 4sU-seq signals at genes that displayed DtoP APA shifts in HSV-1 infected cells involving intronic PAS. IPA: intronic polyadenylation site. (C) PAS-seq and 4sU-seq tracks of TOB2. Red arrows point to the IPA region. (D) 4sU-seq signals at genes that displayed PtoD APA shifts in HSV-1 infected cells. (E) 4sU-seq signals at genes that displayed PtoD APA shifts in HSV-1 infected cells involving intronic PAS. (F) PAS-seq and 4sU-seq tracks of DNAJB6.
Fig 3.
ICP27-dependent and -independent HSV-1-induced APA changes.
(A) A heat map showing the APA index of all the genes that displayed significant APA changes as shown in Fig 1A in mock, wild-type or ΔICP27 HSV-1-infected HeLa cells (8 hpi). Data was scaled by row. (B) A scatter plot showing significant APA differences between mock- and ΔICP27 HSV-1-infected HeLa cells (8 hpi). (C) A Venn diagram showing the overlap between proximal PAS that displayed significant APA changes induced by the wild-type or ΔICP27 HSV-1. (D-F) PAS-seq tracks of mock-, wild-type HSV-1, or ΔICP27 HSV-1-infected HeLa cells at 8 hpi.
Fig 4.
Mechanism of HSV-1-mediated APA regulation.
(A) A scatter plot showing the significant APA changes induced by ICP27 over-expression in HEK293 cells. Color scheme and labeling are similar to Fig 1A. (B) A scatter plot showing the significant APA changes induced by U1 antisense morpholino oligo (AMO)-treatment of HEK293 cells. (C) A heat map of APA index of all genes that displayed significant APA changes induced by HSV-1-infection, ICP27 over-expression, or U1 AMO. Data was scaled by row. (D) PAS-seq tracks for two example genes. (E) GC content at the proximal and distal PAS of genes that displayed significant DtoP shifts induced by ICP27 over-expression (O/E), HSV-1 infection (HSV1), or U1 AMO treatment. (F) ICP27 CLIP signals at the proximal and distal PAS of genes that displayed significant DtoP shifts induced by ICP27 over-expression (O/E), HSV-1 infection (HSV1), or U1 AMO treatment.
Fig 5.
HSV-1-mediated APA regulation and mRNA export.
Average RNA-seq signals for genes that display significant HSV-1-induced APA changes in chromatin, nucleoplasm, and cytoplasm fractions in mock-infected (A) or HSV-1-infected human fibroblast cells (B). P values for the difference in RNA-seq signals at each position between the nuclear and cytoplasmic fractions were calculated using Wilcoxon rank sum test and plotted as a color-coded bar below the plot. The color scheme is the same as Fig 2A. (C) PAS-seq and RNA-seq tracks for HNRNPA2B1 gene. Red arrow points to the PAS-seq signals downstream of the normal TES. (D) PAS-seq signals in the 5kb region downstream of the normal TES in mock- and HSV-1-infected cells. **:P value < 0.05, Wilcox test. (E) Cytoplasmic RNA-seq signals for HSV-1-induced DtoP_intron APA changes. IPA: intronic poly(A) site. (F) PAS-seq and RNA-seq tracks for two example genes. Red arrows point to the IPA.
Fig 6.
HSV-1-induced intronically polyadenylated APA isoforms are translated.
(A) PAS-seq and Ribo-seq tracks for four example genes. Ribo-seq signals within intronic regions are marked by red arrows. (B) Cumulative distribution of the percentage of intronic Ribo-seq read densities (normalized reads per codon) compared to the level in the upstream exon. The dashed line indicates genes exceeding the 5% threshold mentioned in the text. The P value for comparing the pooled read densities in un-infected through 2 hpi vs. 4–8 hpi is shown (two-sided Kolmogorov-Smirnov test) (C) Boxplots showing the distributions of read densities for the 54 genes exceeding the 5% threshold stratified by time point after infection, location with respect to exon-intron boundary and first intronic stop codon, and reading frame of translation. The hinges and whiskers correspond to quartiles and to the most extreme values outside of 1.5 times the inter-quartile range, respectively. The median and outliers are indicated. The y axis is arbitrarily cut at 0.6. P values for comparisons of in-frame and out-of-frame codons are indicated (***, p<0.001; **, p<0.01; *, p<0.05; n.s., not significant at 5% level).
Fig 7.
A model for HSV-1-mediated APA regulation.
In HSV-1-infected cells, ICP27 and other viral factors induce many APA changes and transcription termination defects. Transcripts that extend beyond the normal TES are cleaved and polyadenylated, but are not exported. Truncated transcripts that are polyadenylated at IPA can be exported into the cytoplasm and translated. Please see text for more details.