RNA-binding protein CPEB1 remodels host and viral RNA landscapes

Abstract

Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

Figure 1. Host RNA processing patterns are altered during HCMV infection

(a) Overlap of alternative cassette exon splicing events (top) and alternative polyadenylation (APA) events (bottom) between infected HFFs, NPCs and ECs (n=1 for each condition for each cell type; MOI 5). (b) Numbers and type of infection-altered splicing events in NPCs and HFFs, detected by splicing-sensitive microarrays of Towne-infected HFFs at 72 hpi and TB40E–infected NPCs at 96 hpi. Abbreviations used are: alt, alternative; cass, cassette; mut excl, mutually exclusive; ret, retained. (c) RT-PCR validation of alternative cassette events in mock and HCMV infected HFFs, NPCs, and ECs (3/3 selected events were validated). (d) RNA-seq coverage of PCGF3 and ANKH showing 3′UTR shortening in HCMV infected NPCs (96 hpi) and HFFs. Canonical predicted canonical CPEB1 recognition sites (CPE; specifically, (U)UUUUAU or UUUUAA(U)) are indicated by triangles below the 3’UTRs. (e) qRT-PCR validation of decreased distal 3′UTR usage in PCGF3 and ANKH mRNA transcripts (4/5 targets selected were validated). Error bars = mean +/− standard deviation; n=3 qRT-PCR reaction replicates (f) Identification of altered 3′UTR isoform usage determined by the MISO algorithm in the indicated HCMV viral infection conditions.

Figure 2. RNA binding protein CPEB1 is upregulated in HCMV-infected HFFs, ECs, and NPCs

(a) Heatmap of fold-change (log₂) for host 3′ end processing factors calculated from ratios of RPKMs (normalized to mock, MOI 5). Upregulated = red, and downregulated = green. (b) Immunoblot analysis of CPEB1 upregulation upon HCMV TB40E infection (V, MOI 5) compared to mock uninfected (M) conditions across three cell types. Uncropped blots are shown in Supplementary Data Set 11. (c) Immunoblot analysis of host core 3′ end processing factors. β-actin was used as a loading control. Uncropped blots are shown in Supplementary Data Set 11. (d) Immunofluorescence of HCMV immediate-early (IE) protein CH160 (red) and CPEB1 (C, green) with DAPI (D) in HFFs at 48 hpi (MOI 0.5). Scale bars are 100 µm.

Figure 3. Exogenous CPEB1 expression causes RNA processing changes reminiscent of HCMV infection

(a) Immunoblot of lentivirus-mediated CPEB1 overexpression (OE) in HFFs at 5 days post-transduction. CSTF-77 is shown as a 3′ end processing factor control. Uncropped blots are shown in Supplementary Data Set 11. (b) RNA-seq coverage of the 3′UTR of SYNRG during HCMV and mock infection of HFFs (n=1 per condition). (c) qRT-PCR analysis of distal 3′UTR usage or proximal shift in SYNRG upon lentivirus-mediated CPEB1 OE (compared to GFP OE as a control). Error bars = mean +/− standard deviation; n=3 qRT-PCR reactions. (d) Global analysis for the presence of both a canonical CPE ((U)UUUUAU or UUUUAAU) on either side and PAS (AAUAAA or AUUAAA) upstream of RNA-seq defined 3′ proximal termination sites in increments of 150, 200, and 250 nt. 249 3′UTR shortening events, 78 lengthening events, and 638 control events were considered. Chi-square tests were performed to evaluate significance. *** P-value < 0.0001; * P-value < 0.01. (e) Venn diagram showing overlap between APA changes in CPEB1 OE and HCMV infected HFFs determined from RNA-seq data (n=1 per condition). (f) Venn diagram showing overlap of AS changes in CPEB1 OE and HCMV infected HFFs and NPCs measured by splicing sensitive microarrays (n=1 per condition per cell type). (g) RT-PCR analysis of alternatively spliced cassette exons in MYO18A, SPAG9 and USPL1 mRNA transcripts upon GFP (Control), CPEB1 and CSTF-77 OE compared to HCMV (V) infected HFFs at 48 (V-48) and 96 hpi (V-96).

Figure 4. Genome-wide host alternative splicing remodeling in HCMV infection is replicated by CPEB1 overexpression and corrected by CPEB1 depletion

(a) Hierarchical clustering of alternatively spliced exon indices (as a measure of exon inclusion by RNA-seq in HFFs). Boxed regions highlight a reversal of HCMV infection-related AS (MOI 3). The color bar shows the scale of exon inclusion (+ values) and exclusion (- values) relative to the mean splicing index values when comparing across samples. 472 exons showed reversal as reported in Supplementary Data Set 6 (FDR<0.05, dI >|0.1|). For RNA-seq, n=1 each for mock-NT siRNA, HCMV infected-NT siRNA, HCMV infected-siCPEB1 treated, and GFP OE samples, and n=2 for CPEB1 OE samples. (b) RNA-seq coverage of AS exons (boxed) in ITGA6 and SPAG9 in all conditions in HFF cells (NT is non-targeting siRNA. (c) RT-PCR analysis of AS cassette exons (4/4 selected and validated) of MYO18A, SPAG9, TTC7A, and ITGA6 in either mock (−) or HCMV (+) infected cells treated with either non-targeting (NT) or CPEB1 siRNA.

Figure 5. HCMV infection related host genome-wide 3′UTR shortening and polyA tail lengthening in HCMV infection is reversed by CPEB1 depletion

(a) Heatmap showing 3′UTR shortening (yellow) and lengthening (blue) in Mock vs. HCMV and HCMV vs. siCPEB1 infected cells for 196 genes (Bayes Factor > 10000) common between two RNA-seq datasets (n=1 per condition) (b) RNA-seq coverage of SYNRG 3′UTR in all conditions in HFF cells. Infected cells are indicated by “+”. NT is non-targeting siRNA. (c) qRT-PCR analysis of distal 3′UTR usage in SYNRG and ANKH transcripts in either mock (−) or HCMV (+) infected cells treated with either non-targeting (NT) or CPEB1 siRNA (si-17). Error bars are standard deviation between replicates (n=3 qRT-PCR replicates) and ** P-value < 0.005; * P-value < 0.05 as calculated by Student’s t-test. (d) DAVID gene ontology (GO) shows progressive enrichment of the extracellular exosome category in mock (M) vs. HCMV (V), HCMV (V) vs. siCPEB1 (Si) (+HCMV), and GFP OE vs. CPEB1 OE. (e) Median polyA tail lengths determined by TAIL-seq (n=2 MiSeq runs per condition, datasets from two runs were pooled for final analysis) in HCMV infected HFFs (MOI 3) vs. mock controls and siCPEB1 treated HFFs (+HCMV). (f) Violin plots showing distribution of polyA tail lengths for TOE1 and RAB5C transcripts (mock vs. infection P values <0.0025, 5×10⁻⁵ by Mann Whitney U test, respectively; Infection vs. siCPEB1 P values <0.0015, <2×10⁻⁶ by Mann Whitney U test, respectively).

Figure 6. CPEB1 depletion by siRNA results in shortened polyA tail lengths and decreased protein levels of HCMV late genes

(a) TAIL-seq distributions (n=2 MiSeq runs per condition, datasets from two runs were pooled for final analysis) of median polyA tail lengths for viral genes in HCMV infected HFFs and infected HFFs treated with siCPEB1. (b) Cumulative distribution frequency (CDF) plots of polyA tail length distributions for UL18, UL99, UL83-82-containing and UL54 HCMV transcripts (P values <10⁻⁵, <0.00014, <10⁻²⁰, 0.06, by Mann Whitney U test). (c) UV crosslinking followed by immunoprecipitation coupled with qPCR (CLIP-PCR) for RNA targets bound by CPEB1 and FMRP. n=3 qRT-PCR replicates. (d) Immunoblot analysis of CPEB1 and viral proteins (IEs, UL57, and pp28) in mock (indicated as “−”) or HCMV (TB40E) infected cells (indicated as “+”) treated with either non-targeting (siNT) or four different CPEB1-targeting siRNAs (si-02, si-04, si-17, si-18) at MOIs 0.5 (upper) and 3 (lower). β-actin was used as a loading control. SP stands for smartpool (mixture) of four different siRNAs against CPEB1. Uncropped blots are shown in Supplementary Data Set 11.

Figure 7. CPEB1 depletion rescues cytopathology and attenuates HCMV infection

(a) Phase contrast images of mock or HCMV (TB40E) infected cells treated with either non-targeting (NT) or CPEB1 siRNA (siCPEB1) at low (top; scale bar representing 4×) and high (bottom; scale bar representing 10×) magnifications. Experiment was repeated a total of three times and representative pictures are shown. (b) Productive HCMV viral titers at different time points determined by plaque assay for TB40E infected cells (HCMV TB40E), TB40E infected cells treated with non-targeting (siNT), and TB40E infected cells treated with CPEB1 siRNA (siCPEB1). (c) Immunoblot analysis of CPEB1 and viral proteins (IEs CH160, UL83) in mock (indicated as “−”) and HCMV (TB40E) infected cells (MOI 0.5; indicated as “+”) that are untreated or treated with non-targeting (NT) or CPEB1-targeting siRNA (si17). GAPDH was used as a loading control. Uncropped blots are shown in Supplementary Data Set 11. (d) Our working model summarizes our findings describing HCMV infection in the presence (left) and absence (right) of CPEB1. HCMV infection induces CPEB1 expression which supports certain AS isoforms, shorter 3’UTRs, longer polyA tail lengths and normal productive HCMV titers, whereas CPEB1 depletion leads to preservation of normal 3′UTR lengths, polyA tail lengths, and decreased productive HCMV titers.