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RNA-binding protein CPEB1 remodels host and viral RNA landscapes

Nature Structural & Molecular Biology volume 23, pages 1101–1110 (2016)Cite this article

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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.

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Figure 1: Host RNA-processing patterns are altered during HCMV infection.
Figure 2: RNA-binding protein CPEB1 is upregulated in HCMV-infected HFFs, ECs and NPCs.
Figure 3: Exogenous CPEB1 expression causes RNA-processing changes reminiscent of HCMV infection.
Figure 4: Genome-wide host AS remodeling in HCMV infection is replicated by CPEB1 overexpression and corrected by CPEB1 depletion.
Figure 5: HCMV-infection-related host genome-wide 3′-UTR shortening and poly(A)-tail lengthening in HCMV infection is reversed by CPEB1 depletion.
Figure 6: CPEB1 depletion by siRNA results in shortened poly(A)-tail lengths and decreased protein levels for HCMV late genes.
Figure 7: CPEB1 depletion rescues cytopathology and attenuates HCMV infection.

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Acknowledgements

The authors thank members of the laboratories of G.W.Y. and D.H.S. for critical reading of the manuscript and extend particular acknowledgment to M.T. Lovci (UCSD) for initial assistance with bioinformatics. We thank H. Chang (laboratory of N. Kim, Seoul National University) for help with the Tailseeker algorithm, and S. Azoubel Lima and A. Pasquinelli (UCSD) for sharing their modified TAIL-seq protocol before publication. We especially thank C.S. Morello (UCSD) for assistance with HSV-2 infections, and the laboratory of S.A. Spector (UCSD) for HIV-1-infected materials. This work was partially supported by grants from the National Institutes of Health (HG004659, HG007005 and NS075449 to G.W.Y.) and from the California Institute of Regenerative Medicine (RB3-05219 to G.W.Y. and D.H.S.). T.J.S. and E.C.W. are supported in part by the University of California, San Diego, Genetics Training Program through an institutional training grant from the National Institute of General Medical Sciences, T32 GM008666. E.C.W. is supported as an NSF Graduate Research Fellow. R.B. is supported as a Myotonic Dystrophy Foundation postdoctoral fellow. G.W.Y. is supported as an Alfred P. Sloan Research Fellow.

Author information

Author notes

  1. Ranjan Batra and Thomas J Stark: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA

    Ranjan Batra, Thomas J Stark, Alex E. Clark, Jean-Philippe Belzile, Emily C Wheeler, Brian A Yee, Hui Huang, Chelsea Gelboin-Burkhart, Stephanie C Huelga, Stefan Aigner, Brett T Roberts, Tomas J Bos, Shashank Sathe, Deborah H Spector & Gene W Yeo

  2. Stem Cell Program, University of California at San Diego, La Jolla, California, USA

    Ranjan Batra, Thomas J Stark, Emily C Wheeler, Brian A Yee, Hui Huang, Chelsea Gelboin-Burkhart, Stephanie C Huelga, Stefan Aigner, Brett T Roberts, Tomas J Bos, Shashank Sathe & Gene W Yeo

  3. Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA

    Ranjan Batra, Thomas J Stark, Emily C Wheeler, Brian A Yee, Hui Huang, Chelsea Gelboin-Burkhart, Stephanie C Huelga, Stefan Aigner, Brett T Roberts, Tomas J Bos, Shashank Sathe & Gene W Yeo

  4. Division of Biological Sciences, University of California at San Diego, La Jolla, California, USA

    Thomas J Stark, Alex E. Clark & Deborah H Spector

  5. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California, USA

    Alex E. Clark, Jean-Philippe Belzile & Deborah H Spector

  6. Department of Molecular, RNA Center, Cell and Developmental Biology, Sinsheimer Labs, University of California, Santa Cruz, Santa Cruz, California, USA

    John Paul Donohue & Manuel Ares Jr

  7. Ionis Pharmaceuticals, Carlsbad, California, USA

    Frank Rigo

  8. Molecular Engineering Laboratory, A*STAR, Singapore

    Gene W Yeo

  9. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Gene W Yeo

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Contributions

R.B., T.J.S., D.H.S. and G.W.Y. designed the study and wrote the manuscript; A.E.C. and J.-P.B. maintained HCMV viral stocks, measured titers and performed HCMV infections; R.B., T.J.S. and A.E.C. performed siRNA treatments. T.J.S., A.E.C. and R.B. performed western blots. R.B. performed immunofluorescence and microscopy. T.J.S. and T.J.B. made lentivirus preparations. R.B., T.J.S. and E.C.W. made RNA-seq libraries. R.B., T.J.S., S.C.H., B.A.Y. and S.S. performed bioinformatics analysis. R.B. and T.J.S. performed RT–PCR splicing and APA assays. H.H. and R.B. prepared TAIL-seq libraries, as overseen by S.A. C.G.-B. performed microarray hybridizations. S.C.H., J.P.D. and M.A. performed microarray analysis. R.B., T.J.S. and B.T.R. performed RT–PCR splicing and APA assays. F.R. provided the antisense oligonucleotides.

Corresponding author

Correspondence to Gene W Yeo.

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Competing interests

F.R. is a paid employee of Ionis Pharmaceuticals, which provided ASO reagents.

Integrated supplementary information

Supplementary Figure 1 Viral gene expression differs among cell types.

(a) The percent of mapped sequencing reads between the human and viral genomes for HFFs, ECs and NPCs at 48 and 96 h post HCMV TB40E-infection (hpi) from RNA-seq data (n=1 per condition). (b) Pearson correlation matrix of HCMV gene expression values in HFFs, ECs, and NPCs at 48 and 96 hpi. (c) Scatter plot comparisons of HCMV (TB40E) gene levels (Log2 RPKM). Significantly under-represented genes in NPCs at 96 hpi are highlighted in green. (d) Comparison of the highest fold-change differences between 96 hpi NPCs and 96 hpi TB40E HFFs and ECs (genes labeled green in c). Associated temporal gene classes are indicated. D.E. is Delayed Early.

Supplementary Figure 2 Host alternative polyadenylation changes occur during herpesvirus infections.

(a) RNA-seq coverage of MARCH6 3′UTR in HCMV infected NPCs (96 hpi) and HFFs (96 hpi). Canonical predicted CPEB1 recognition sites (CPE; specifically, (U)UUUUAU or UUUUAAU) are indicated by triangles below the 3’UTR. (b) qRT-PCR validation of decreased distal 3′UTR usage in MARCH6 mRNA transcript in HCMV infection (96 hpi, mean +/- standard deviation; n=3). (c) Global distribution of changes in 3′UTR length among shortening events during HCMV infection in three cell types. (d) Bar plot showing altered 3′UTR isoform usage determined by the MISO algorithm 8 hours post HSV-2 infection in HFFs and HIV-1 infected T-cells (left). Venn diagram showing overlap in APA events in HCMV and HSV-2 infections (right). (e) RNA-seq coverage of PCGF3 3′UTR in HSV-2 infected HFFs (8 hpi). (f) qPCR validation of 3′UTR shortening in PCGF3 and ANKH. Error bars = mean +/- standard deviation; n = 3 replicates. (g) RNA-seq coverage of PICALM showing alternatively spliced exon (exclusion) in HSV-2 infection 8 hpi.

Supplementary Figure 3 HCMV infection causes host gene-expression changes.

(a) Heatmap of host gene expression changes (all genes) in HFFs, ECs, and NPCs at 48 and 96 hpi with HCMV TB40E. (b) Venn diagram showing dynamics of host gene expression changes from 48 to 96 hpi with HCMV TB40E. The grey circles are the host genes with altered expression between mock vs. HCMV at 48hpi and black circles represent genes altered at 96 hpi in respective cell types. (c) Venn diagrams of differentially expressed host genes in HCMV TB40E infected HFFs (black), ECs (green), and NPCs (red) at 48 and 96 hpi. (d) qRT-PCR showing CPEB1 upregulation in NPC cells (differentiated from HUES9 and H9 human embryonic stem cells) at 96 hpi with HCMV TB40E. Error bars are mean +/- STD; n=3. (e) Immunoblot showing CPEB1 upregulation in NPCs (from H9) 96 hpi with HCMV TB40E. (f) Hierarchical clustering of log2 RPKM values of all known and predicted RNA binding proteins (RBPs). CPEB1 is shown in the box. Color bar shows log2 scale. Raw values and visualization are also shown in Supplementary Data Set 4b.

Supplementary Figure 4 CPEB1 is specifically upregulated during HCMV infection.

(a) Immunoblot showing CPEB4 protein levels in mock and TB40E HCMV infection of HFFs at 48 and 96 hpi. Beta actin was used as a loading control. (b) Immunoblot showing CPEB2 and CPEB4 protein levels in mock and TB40E infected HFFs at 48hpi. GAPDH was used as a loading control. (c) Immunoblot analysis of hnRNP proteins and core 3′UTR machinery factors in mock (M) and HCMV (V) treated HFFs (MOI 5) at 48 hpi. β-actin was used as a loading control. (d) Immunoblot analysis of CSTF-64 at 24 hours post mock treatment or HCMV infection in presence or absence of CSTF-64 shRNA treatment. (e) qRT-PCR analysis of distal over proximal PCGF3 3′UTRs at 24 and 72 hpi with HCMV after control or CSTF-64 shRNA treatment. Error bars are mean +/- STD; n=3 (f) qPCR showing fold change over mock mRNA levels for CPEB1 in mock, interferon-gamma (IFN-g), UV-inactivated TB40E, and live TB40E HCMV treated HFFs. (g) RT-PCR of exon exclusion levels in SPAG9 and ITGA6 in HCMV treated HFFs compared to IFN-g, UV-inactivated virus, and mock treatment. (h) Immunoblot of CPEB1 in mock or HCMV (TB40E) infected HFFs treated with non-targeting siRNA (NT1) and two different CPEB1 siRNAs (si1 and si2) at 48 hpi. CH160 antibody was used to identify the immediate early HCMV infection markers IE72 and IE86. β-actin was used as a loading control. The antibody used recognized CPEB1 as two bands and both bands were depleted with siRNA treatments. (i) Bar chart showing cell viability (measured by presto blue reagent) in uninfected cells that are untreated or treated with NT siRNA or siCPEB1, and in HCMV infected cells at 48 hpi that are treated with NT siRNA or siCPEB1 siRNA. Error bars show standard deviation between three replicates.

Supplementary Figure 5 CPEB1-related alternative polyadenylation changes in HCMV infection and their enrichment in extracellular exosomal genes.

(a) Scatter plot of mean distal APA site indices (psi values or posterior mean values in Supplementary Data Set 3) of siNT in mock (x-axis) vs. siNT in HCMV-infected (y-axis) cells. Red dots show 3′UTR shortening (76%) and blue dots show 3′UTR lengthening (24%) during HCMV infection (Colored dots have Bayes Factor > 10000); this color codes also applies to (b) and (c). (b) Scatter plot of mean distal APA site indices in GFP OE (x-axis) vs. CPEB1 OE (y-axis). Data shows 3′UTR shortening (77%) and 3′UTR lengthening (23%) with CPEB1 OE. (c) Venn diagram of APA changes in CPEB1 OE and HCMV (+) HFFs measured by RNA-seq. (d) Scatter plot of mean distal APA site indices in siNT in HCMV (x-axis) vs. siCPEB1 in HCMV-infected (y-axis) cells. Data shows 3′UTR shortening (39%) and 3′UTR lengthening (61%) with CPEB1 depletion in HCMV infection. (e) RNA-seq coverage of RPS6KA3 (Ribosomal S6 protein kinase associated with growth) showing 3 ‘UTR shortening (delta-psi = -0.56, Bayes Factor >1012) during infection and reversal with siCPEB1 (delta-psi = 0.15, Bayes Factor = ~5 x 105). 3′UTR shortening is also observed in CPEB1 OE compared with GFP OE (delta-psi = -0.39, Bayes Factor >1012). Known APA sites, target scan (TS) miRNA binding sites and 100-vertebrate nucleotide conservation tracks from UCSC are shown below. (f) Venn diagram showing overlap between APA changes in siNT in mock vs. siNT in HCMV and siNT in HCMV vs. siCPEB1 in HCMV-infection at 48hpi in HFFs. 196 genes are reversed (lengthened) in siCPEB1 compared to HCMV. (g) qRT-PCR analysis of distal 3′UTR usage in PCGF3 and ANKH in mock, HCMV infection (48 hpi), GFP OE, or CPEB1 OE. Error bars represent standard deviation between three replicates. We validated total 3/4 targets tested based on targets common in HCMV infection and CPEB1 OE. (h) DAVID gene ontology (GO) analysis shows progressive enrichment of extracellular exosome category in APA altered genes in siNT in mock vs. siNT in HCMV, siNT in HCMV vs. siCPEB1 in HCMV-infected, and GFP OE vs. CPEB1 OE. Y- axis is –log2 P value with FDR correction applied.

Supplementary Figure 6 siRNA- and antisense oligonucleotide–mediated CPEB1 depletion affects HCMV genes and cell morphology.

(a) PolyA tail length assessment of spike-ins with known tail lengths (0, 8, 16, 32, 64, 128 As) show accurate tail length determination by the Tailseeker2 program. (b) Bar plot showing enriched GO categories in genes decreased in polyA tail lengths post siRNA-mediated CPEB1 depletion in HCMV infected HFFs. (c) Phase contrast images of HFFs under the following conditions: NT siRNA-Mock; NT siRNA-HCMV; CPEB1 siRNA-HCMV; UL99 (pp28 protein) overexpression-CPEB1 siRNA-HCMV; and CPEB1 overexpression-CPEB1 siRNA-HCMV (top). Phase contrast and GFP overlay of UL99-overexpression and CPEB1 siRNA-HCMV, and CPEB1 overexpression-CPEB1 siRNA-HCMV HFFs (bottom) at lower (left) and higher (right) magnifications. GFP is present in the same transcript as UL99 or CPEB1 and translated through an internal ribosome entry site (IRES). CPEB1-GFP lenti is codon optimized and resistant to CPEB1 siRNA. (d) RT-PCR using primers designed on the flanking exons shows activity of 19 different ASOs (including a control ASO in lane 1) designed to target exon 5 of CPEB1 pre-mRNA. ASOs were designed to block the 5’ splice site (5’ ss) or 3’ splice site (ss) of the specified exons. Boxed ASOs 2 and 14 were successful in causing exon skipping. Use of ASO 2 and 14 lead to a decrease in overall CPEB1 mRNA levels. (e) Quantitative RT-PCR with primers designed to interrogate the 3′UTR of CPEB1, showing CPEB1 levels during infection and their reduction by different ASOs and their combinations. Combination of ASOs 14 and 2 (blocking both 5’ and 3’ ss of exon 5) was the most effective in reducing CPEB1 mRNA levels. The error bars are standard error around the mean of the replicates. (f) Immunoblot showing CPEB1, UL57, and pp28 levels in mock, HCMV infected non-targeting (NT) and smart pool (SP) siRNA treated, and ASO-treated HFFs. GAPDH was used as a loading control and UV-treated HCMV was used as a negative control. RT-PCR in the lower panel shows SPAG9 AS for respective samples.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 1117 kb)

Supplementary Data Set 1

HCMV gene expression calculations (XLSX 21 kb)

Supplementary Data Set 2

All significantly changing host alternative splicing events from microarrays (XLSX 489 kb)

Supplementary Data Set 3

Significantly changing alternative host polyadenylation events in 2 or more cell types (S3a), Mock vs. HCMV (S3b), HCMV vs. siCPEB1 (S3c), GFP OE vs. CPEB1 OE (S3d), and Mock vs. HSV-2 (S3e) (XLSX 335 kb)

Supplementary Data Set 4

Significant host gene expression changes and clustering groups. (XLSX 3059 kb)

Supplementary Data Set 5

Alternative cassette exon splicing event changes common to CPEB1 OE and HCMV infected cells (XLSX 122 kb)

Supplementary Data Set 6

Significant host gene alternative cassette splicing changes upon CPEB1 KD and CPEB1 OE from RNA-seq (XLSX 1049 kb)

Supplementary Data Set 7

Significant Gene Ontology (GO) enriched categories for APA altered genes (XLSX 46 kb)

Supplementary Data Set 8

Poly(A) tail lengths analyzed by TAIL-seq (XLSX 886 kb)

Supplementary Data Set 9

Antisense oligonucleotides (ASOs) targeting CPEB1 (XLSX 10 kb)

Supplementary Data Set 10

Uncropped immunoblots (PDF 33685 kb)

Supplementary Data Set 11

Primer sequences for RT–PCR and qPCR (XLS 30 kb)

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Batra, R., Stark, T., Clark, A. et al. RNA-binding protein CPEB1 remodels host and viral RNA landscapes. Nat Struct Mol Biol 23, 1101–1110 (2016). https://doi.org/10.1038/nsmb.3310

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