© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. to to G.W.Y. ( School of Medicine, National University of Singapore, Singapore. California, USA. La Jolla, California, USA. University of California at San Diego, La Jolla, California, USA. Diego, La Jolla, California, USA. 1 unexplored. largely are infection been studied have splicing and expression gene viral although and infection, lytic such wide-ranging changes may be responsible for progressive HCMV of capable factors RNA-regulatory Host processing. RNA cellular of for rapid alteration of the host cellular environment is the modulation occurs levels mRNA host in decrease overall synthesis protein and occurs transcription host of shutdown complete a ment must be poised for viral replication, and for largely some herpesviruses, remains transcriptomes environ cellular the infection, unclear. In of host stages herpesvirus early and viral between and interaction response the but types, cell of variety a infects HCMV tion. options are limited, and there is currently no vaccine to prevent infec been associated with glioblastoma and atherosclerosis and newborns in defects severe in disease immunocompromised individuals neurological severe cause can Infection deficits. developmental have in year United the pervasive each States adulthood nearly 100% of in individuals the United States by the time they reach affect health worldwide. HCMV, a family herpesvirus member, infects and substantially prevalent are highly infections Human herpesvirus of occurrence also related poly(A)-tail of gene in Here, Host H Deborah Spector Brett T Roberts Brian A Yee Ranjan Batra RNA landscapes RNA-binding protein CPEB1 remodels host and viral nature structural & molecular biology molecular & structural nature Received 1 August; accepted 22 September; published online 24 October 2016; Department Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.

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© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. Fig. 2c were generally shortened ( transcripts affected of lengths APA,For the ECs. and HFFs or NPCs Supplementary Fig. 2a (3 (exons in ( types 3 shortened in resulted events exons detected in the RNA-seq data. Interestingly, the majority of these algorithm (MISO) isoformsof mixture the used we transcripts, host in changes programASestablished similaris byHCMVinfection.To studyAPA ( alternative-cassetteNPC the eventsoverlapped changesHFFs with in HFFs and NPCs events( RNAalteredsignificantlyhostsplicingmicroarrays,found >2,000 we Affymetrix splicing-sensitive Using infection. HCMV of result a as times ( infection early the during at lines cell three the in similar was expression mRNA model NPC this in infection nonprogressive Data Set 1 96 at ECs and HFFs hpi, ( but not in NPCs in genes viral in changes expression marked Fig. 1b with infection time points in HFF, EC and NPC cells ( of infection viral degrees varying that support types cell infected are commonly These TB40E (multiplicity of infection (MOI) of 5; isolate HCMV clinical the with (hpi) postinfection h 96 and 48 both at (NPCs) cells progenitor neural cell–derived (ES) stem embryonic human and (ECs) cells endothelial aortic human (HFFs), fibroblasts poly(A) generated we genes, HCMV and host of patterns expression mRNA Toassess Host RESULTS infection. HCMV lytic full-blown for necessary transcripts viral and host modulating for required is CPEB1 that demonstrate of results levels Our titers. viral protein productive decreased and decreased proteins viral late substrates, CPEB1-bound in lengths changes. Strikingly, decreased CPEB1 led to shortened viral poly(A)-tail host AS, APA of of a infection-related and reversal large cPAfraction to the led infection HCMV during of CPEB1 depletion Additionally, APApatterns. and AS host infection-related recapitulated infection of HCMV absence the in HCMV of CPEB1 expression Exogenous during infection. upregulated is CPEB1 RBP the that discovered we Unexpectedly, changes. RNA-processing and expression gene viral host and to leads widespread that HCMV infection revealed analyses study,transcriptome-wide this In understood. poorly remains tions infec virus during transcriptomes of or host viral modulation in the control both AS and APA ogeneous nuclear (HNRNPs) ribonucleoproteins have shown been to of RBPs such as muscleblind-like (MBNL), NOVA, RBFOX and heter quently determine cellular fates in differentiation and disease. Families conse and they in cells, RNA and the shaping landscapes processing infections. viral and disease human in determined been not have lengths poly(A)-tail in changes transcriptome-wide However, s e l c i t r a  at 8 hpi and analyzed APA with the MISO algorithm ( Fig. 1 Fig. ′ RNA-binding proteins (RBPs) are responsible for regulating RNA regulating for responsible are (RBPs) proteins RNA-binding Next, we evaluated alterations in host-cell RNA-processing pathways UTRs in

RNA ). We also generated RNA-seq data from HFFs infected with HSV-2 Fig.1 a ; based on annotated HCMV open reading frames), we observed Fig. 1a Fig. and 2 USPL1 ). ). This finding was consistent with previous observations of 7

to survey tandem 3 processing PCGF3 a Supplementary Data Set 2 Set Data Supplementary and , b 2 , and 3 8 CAST 0 . Although NPCs exhibited fewer AS changes, half of Supplementary3 Data Set + . Indeed, although viral gene correlated . viral expression although Indeed, , RNA-seq libraries from primary human foreskin from primary RNA-seq libraries ANKH Supplementary Data Set 2 SupplementarySet Data Supplementary Fig. 1b Fig. Supplementary Supplementary Fig. 1c Fig. Supplementary ,

b and is ) events that were specific to either the infected 17

Fig. 1 extensively , 19 and MYO18A , 26– ′ ′ -UTR events and alternative 3 UTRs after infection in the three cell cell three the in infection after UTRs f MARCH6 ) by 1–5 kb by 96 hpi ( 2 9 . . However, the involvement of RBPs

transcripts in altered ), thus demonstrating that a that demonstrating thus ), transcripts in Supplementary Fig. Supplementary 1a 3 ). We). validated ASboth

0 , ). in d . Nonetheless, HCMV HCMV . Nonetheless, and

) in HCMV-infectedin ) HCMV Fig. 1 Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary Fig. 1d

infection c ) and APA advance online publication online advance ′ -terminal , e and ). ). - - -

altered by the herpesvirus family members HCMV and HSV-2. APAconcludedandthatAS RNAhost transcriptsin aredramatically cells infected with the RNA virus HIV-1 ( ( Fig. 2e Fig. right) in host ( transcripts events including APA HCMV with common were events, ening Data Set 3e infection conditions. Source Data for usage determined by the MISO algorithm in the indicated HCMV viral ( (4/5 targets selected were validated). Data are shown as mean decreased distal 3 are indicated by triangles below the 3 CPEB1-recognition sites (CPEs, specifically (U)UUUUAU or UUUUAA(U)), shortening in HCMV-infected NPCs (96 hpi) and HFFs. Predicted canonical validated). ( mock- and HCMV-infected HFFs, NPCs and ECs (3/3 selected events were ret, retained. ( at 96 hpi. Alt, alternative; cass, cassette; mut excl, mutually exclusive; microarrays of Towne-infected HFFs at 72 hpi and TB40E-infected NPCs altered splicing events in NPCs and HFFs, detected by splicing-sensitive condition for each cell type; MOI 5). ( (bottom) among infected HFFs, NPCs and ECs ( ( Figure 1 n a Supplementary Fig. 2g a e d c ) Overlap of alternative cassette-exon splicing events (top) and APA events = 3 qRT–PCR reactions). ( Usage (distal/proximal) NPC HFF 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 601 275 295 157 24 - - - - 48 , pA 48

f Mock Mock Mock HCMV HCMV PCGF3 HFF ). We also found AS changes in transcripts such as such transcripts in changes AS foundWe also ). Host RNA-processing patterns are altered during HCMV infection. 96 72 CPE 48 d 1,828 ). We found 278 altered events (173 (~62%) 3 58 ) RNA-seq coverage of 96 108 PCGF3 c 96 Supplementary Fig. 2d Fig. Supplementary ) RT–PCR validation of alternative-cassette events in 34 | 741 81 24 TATAAA 48 676 ′ -UTR usage in Mock 96 83 48 341

Towne HFF NPC 560 48 nature structural & molecular biology molecular & structural nature 2 kb 72 96 HCMV ). However, we did not find APA changes in T 96 0.2 0.4 0.6 0.8 1.0 1.2 1.4 EC HFF NPC f 0 ) Identification of altered 3 48 48 Mock HFF 96 PCGF3 96 b 48

PCGF3 Number of events TB40 4,000 1,000 2,000 3,000 48 e ANKH ′ 96 b UTRs. ( | ANKH and ) Numbers and types of infection- AATAAA 113 825 548 0 96 E and 94 48 ) out of which 51 events (18%) events 51 which of out ) - - - - and Al f 3 48 96 are available online. Mock ′ t NP and Supplementary Fig. 2d ANKH 48 e Mock Mock HCMV HCMV 96 C Al ) qRT–PCR validation of n 5 ANKH 96 isoforms EC ′ upeetr Fg 2d Fig. Supplementary t = 1 experiment for each UT PCGF3 TB40E 48 cass R Al mRNA transcripts f 2 kb t MISO 3′ events 96 , showing 3 1,000 1,500 end Al ANKH 50 HFF 48 | ′ 48 t AATGAA -UTR isoform 0 0 Mock ( CP star

Supplementary HFF 96 Al E 96 h 3 NP ′ t -UTR status t Extended ′ Shortened

± EC 48 C TB40E -UTR short 48 h excl Mu s.e.m. |

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© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. infection, we treated cells with UV-inactivated HCMV or interferon interferon or HCMV UV-inactivated with cells treated we infection, ( 3 candidate affect not did lation ( HFFs infected in protein CSTF-64 the of is not associated with 3 types ( cell three all in isoform CSTF-77 longer a of induction an was there ( types cell across altered were not consistently members family CPEB other HNRNP family members (HNRNPH, HNRNPM and HNRNPU) and proteins), CPSF100 and CPSF160 CF-Im68, CSTF-77, CSTF-64, the levels of most of the known mediators of 3 3d Fig. Supplementary and ECs both in NPCs, by 96 hpi) and protein 30-fold levels during HCMV ( infection over and HFFs in (six-fold mRNA the at types cell all in RBP upregulated consistently and dramatically most 4 Set Data Supplementary 4 Set ( infection. HCMV during genes of RNA-seq) (by abundance mRNA the Weevaluated factors. RNA-regulatory common more or one of involvement the 3 of shortening The CPEB 100 bars, Scale 0.5). (MOI hpi 48 at HFFs in (D) stain nuclear in shown ( types. cell three across conditions ( downregulated. green, MOI 5). upregulated; Red, to infection, mock (normalized (RPKM) reads mapped per million transcript of per kilobase reads ratios from calculated factors 2 Figure nature structural & molecular biology molecular & structural nature (IFN- gamma Supplementary Fig. 4d Fig. Supplementary ), focusing on host RBPs known to regulate APA ( APA regulate to known RBPs host on focusing ), 1 Fig. Fig. 2 Fig. 2 Fig.

is RNA-binding protein CPEB1 is upregulated in CPEB1 is protein HCMV-infected upregulated RNA-binding HFFs, ECs and NPCs. ( Supplementary Data Set 10 Set Data Supplementary

consistently PAPOLG PAPOLA a PABPC1 PPP1CB PABPN1 PPP1CA NUDT21 CSTF2T SYMPK WDR33 RBBP6 CPEB1 CPEB2 CPEB4 CPEB3 FIP1L1 CPSF6 CPSF1 CPSF7 CPSF2 CPSF3 CPSF4 c CSTF1 PCF11 CSTF2 CSTF3 c CLP1 ), ), but, according to a previous report, the longer isoform and and γ ), neither of which induced CPEB1 upregulation upregulation CPEB1 induced which of neither ), Supplementary Figs. 3f 3f Figs. Supplementary Supplementary Fig. 3 Fig. Supplementary 48 ′ HFF UTRs among the different cell types suggested suggested types cell different the among UTRs h b ′

) Immunoblot analysis of CPEB1 upregulation after HCMV TB40E infection (V; MOI 5) compared with mock-infected (M) (M) mock-infected with (V; compared 5) MOI infection TB40E HCMV after upregulation CPEB1 of analysis ) Immunoblot

-UTR shortening 96 upregulated – h f , e and 48 EC ). To demonstrate specificity for HCMV HCMV for specificity Todemonstrate ). h ). Unexpectedly, CPEB1 emerged as the the as emerged CPEB1 Unexpectedly, ). 96

Supplementary Data Set 3e Set Data Supplementary h

48 NPC c ′ ) Immunoblot analysis of host core 3 core host of analysis ) Immunoblot h -UTR shortening events in HFFs HFFs in events shortening -UTR . . ( 96

d

during h ) Immunofluorescence of HCMV immediate early (IE) protein CH160 (red) and CPEB1 (C, green) with DAPI DAPI with green) (C, CPEB1 and (red) CH160 protein (IE) early immediate HCMV of ) Immunofluorescence

log RPKM fold change over mock

3 2 –5 0 5 1 ′ . We observed upregulation and and -end -end formation (including Fig. 2 Fig.

and c d b M HCMV V Supplementary Data Data Supplementary

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-actin -actin c I infection m ). Interestingly, Interestingly, ). 68 Fig. 2 Fig. µ 48 48 ). ). Protein m. Fig. Fig. 2 M M a HFF 96 and and 96 HFF CPEB1 (C) b s ′ - -end-processing factors. factors. -end-processing , s 48 48 V that ~50% of isoforms that shortened during infection exhibited exhibited infection during shortened that isoforms of ~50% that found we Indeed, sites. cleavage transcripts alternative affected surrounding regions for in reported been has elements these in ment the recently proposed role of CPEB1 in RNA processing RNA in CPEB1 of role proposed recently the for sufficient are alone elements sequence these whether unknown 3 to CPEB1 recruit to known (PAS) are signal polyadenylation a to proximity in (CPE) sequences ( infection during shift toward utilization of proximal poly(A) sites in transcripts affected substantial a caused cells uninfected in (OE) overexpression CPEB1 HFFs noninfected in ( CPEB1 of isoform full-length the expressed RNA- the for responsible during we HCMV changes processing indeed infection, ectopically observed is CPEB1 whether evaluate To CPEB types. cell three all in infection after upregulated robustly and HCMV-infected Thus, cells. we concluded that CPEB1 is consistently ( by immunofluorescence at hpi, 48 as detected antibody) CH160 IE2; and (IE1 proteins viral early immediate major the with marked 0.5) (MOI cells infected the in observed was upregulation CPEB1 level, and ( V Fig. Fig. 2 Supplementary Fig. 4f Fig. Supplementary i. 3 Fig. 96 96 ITGA6 1 48 d a 48 expression ), ), and CPEB1 was present in both the nuclei and ofcytoplasm ) and subjected the cells to RNA-seq analysis. Strikingly, Strikingly, analysis. RNA-seq to cells the subjected and ) V M

M CH160 (IE) 96 transcripts ( transcripts 96 a ECs ECs ) ) Heat map of fold (log change 48 48

β causes SYNRG V -actin is a loading control. Uncropped blots are are blots Uncropped control. a loading is -actin 96 96 ) or caused HCMV-related AS in the the in AS HCMV-related caused or ) Supplementary Fig. 4g Fig. Supplementary 48

changes 48 in in V M M C/IE ′ -UTR regions -UTR 96 i. 3b Fig. 96 NPCs NPCs

reminiscent 48 48 V , c . osnu UUUUUAU Consensus ). 96 96 2 ) ) for host 3 2 hpi D/C/IE hpi 2 . Although it remains remains it Although .

of ). At the subcellular subcellular the At ). s e l c i t r a

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2 infection , enrich , SPAG9  -

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. ited 3 ited 5f Fig. Supplementary ( KD CPEB1 after reversed completely or partially either 3 Set ( cells 3 observed we ( APA changes observed the in ( sion ( infection ing AS. HCMV-infection-related reverses KD reversion after CPEB1 depletion during Thus, virus infection. CPEB1 and and Integrated Discovery (DAVID) Discovery Integrated and Visualization for Annotation, Database with analysis (GO) ontology ( effect this have not did known to 3 affect ( ANKH ( KD CPEB1 after cells infected in UTRs vidual AS events in the transcripts indi of Evaluation events. these of regulation the to RBPs other of contribution the or CPEB1 of KD incomplete of result the probably Data Set 6 Supplementary reversed <0.05; rate (FDR) completely discovery (false depletion or CPEB1 after partially either were infection HCMV ing dur established changes AS the of 1,150) of (472 41% Furthermore, ( overex HFFs HCMV-infected to HFFs closer clustered CPEB1 pressing uninfected Notably, cells. mock-infected to similar were cells) (siCPEB1 CPEB1 of depleted HFFs HCMV-infected that demonstrated infection HCMV during altered exons of basis the on CPEB1 (CPEB1 OE1 and and CPEB1 OE2). OE) Hierarchical clustering of AS changes (GFP GFP overexpressing HFFs in and hpi, 48 at siRNA CPEB1-targeting and siRNA control (NT) nontargeting with treated cells HCMV-infected and mock- in RNA-seq performed we the compare of transcriptomes HCMV-infected to and CPEB1-overexpressing HFFs, and HFFs of infection HCMV during changes ing 4h Fig. ( viability cell decreasing without depletion CPEB1 >80–90% achieved and (siRNAs) RNAs interfering short different down (KD) of CPEB1 in HFFs 24 h before (MOI infection 3) by using we RNA-interference-mediated these possibilities, performed knock tion or may be of a Toitself. effect downstream the infection examine program may be important in the HCMV full establishing lytic infec The upregulation of CPEB1 and alterations in the host RNA-processing CPEB HFFs. in infection HCMV during changes RNA-processing of the fraction for a large is responsible CPEB1 that in changes no caused yet in Notably, CPEB1 OE, but not CSTF-77 AS OE, similar caused changes infected between in OE CPEB1 HFFs and HFFs without occurred consistent infection. NPCs also were that microarrays) ing-sensitive and of the APA by changes (detected RNA-seq) and 43% (276/645, (128/473; 27% that showed ( infection during above a that of two-fold unchanged value that remained the isoforms 3 the proximal surrounding CPE-PAS co-occurrences s e l c i t r a  (STRING) Genes Neighboring of Instances Supplementary Fig. 5g Supplementary

Global APA analysis primarily showed 3 showed primarily analysis APA Global MYO18A Supplementary Data Set 5 Supplementary MYO18A ). 196 (of 473; 41%) APA changes in HCMV infection were were infection HCMV in changes APA 41%) 473; (of 196 ). ′ 1 Supplementary Fig. 5b Fig. Supplementary Fig. 5 Fig. -UTR shortening during infection maintained their longer 3 longer their maintained infection during shortening -UTR transcripts showed 3 showed transcripts , loss i ). ). Toof to CPEB1 RNA-process contribution the determine

and a reverses , , ( Supplementary Fig. 5a Fig. Supplementary ′ Supplementary Fig. 5d Fig. Supplementary -UTR lengthening after CPEB1 depletion in infected infected in depletion CPEB1 after lengthening -UTR Fig. Fig. 4 SPAG9 ′ cleavage and of polyadenylation mRNAsprecursor Fig. 3 Fig. c

HCMV-infection-related ) ) with RNA-seq and RT–PCR assays confirmed that resembled changes after HCMV infection ). ). d ). ). Importantly, of depletion a CSTF64, factor Supplementary Fig. 4e Fig. Supplementary ). Additionally, genome-wide comparisons comparisons genome-wide Additionally, ). SYNRG USPL1 ). The partial ). reversion of The partial AS patterns was Fig. 3 Fig. ′ -UTR shortening in CPEB1 OE HFFs HFFs OE CPEB1 in shortening -UTR ), and there was a significant overlap overlap significant a was there and ), Supplementary Fig. 5c Fig. Supplementary ) ) of the AS changes by (detected splic ( and and e Fig. 3 Fig. ITGA6 and and 3 3 and Search Tool for Recurring Tool Recurring Search for and ) and with CPEB1 overexpres CPEB1 with and ) ANKH , e Supplementary Data Set 3 Set Data Supplementary g and and , , ). Therefore, we concluded concluded we Therefore, ). Fig. 5b Fig. SPAG9 ′ transcripts that exhib that transcripts -UTR shortening dur shortening -UTR 3 Supplementary Data Data Supplementary 4

f ee wt APA with genes of ). Interestingly, gene gene Interestingly, ). RNA , ( c ), and and ), Fig. Fig. 4b Supplementary Supplementary

′ changes ends (250 nt), ends (250 ). In contrast, contrast, In ). PCGF3 Fig. 5 Fig. Fig. 4 Fig. , c advance online publication online advance ), ), Fig. 4 Fig. TTC7A Fig. Fig. 3 a a and and and and and a ). ). ------) f ′

HCMV-infection-related 3 HCMV-infection-related 5h Fig. Supplementary infected HFFs infected events, P were further enriched in siCPEB1 cells (reversal or lengthening events; ( ratus’ exosome’ ( ‘extracellular GO terms ( changes for data Source (V-96). hpi 96 and (V-48) 48 at HFFs (V)-infected HCMV with compared CSTF-77, or CPEB1 MYO18A ( type). cell ( microarrays splicing-sensitive by measured NPCs, and HFFs HCMV-infected and OE CPEB1 in changes AS ( condition). per experiment RNA-seq ( data RNA-seq from determined HFFs, HCMV-infected and OE ( *** significance. evaluate to performed were tests 78 lengthening events and 638 control events were 3 considered, 249 nt. 250 and 200 150, of andincrements chi-square 3 RNA-seq-defined of upstream AUUAAA) or a PAS and (AAUAAA side either on UUUUAAU) or ((U)UUUUAU CPE ( reactions). PCR mean as shown are Data a control). as OE GFP with in shift proximal ( condition). per experiment of UTR in shown a 3 as shown is CSTF-77 5 d post-transduction. at HFFs in (OE) overexpression CPEB1 ( infection. HCMV of reminiscent 3 Figure e CSTF-77 28 × 10 × 2.87 = d a ) Venn diagram showing overlap between APA changes in CPEB1 CPEB1 in APA changes between overlap showing ) Venn diagram f CPEB1 CPEB1 eas ctpamc PB lvl ae lo ihr n HCMV- in higher also are levels CPEB1 cytoplasmic Because β AS CPEB1 OE O/E

% of total events - actin 10 20 30 40 50 0 914 Proximal SYNRG P ±

P , , 150 bp = 1.3 × 10 × 1.3 = Exogenous CPEB1 expression causes RNA-processing changes changes RNA-processing causes expression CPEB1 Exogenous Supplementary Data Set 10 Set Data Supplementary CPE +PAS *** = 5.7 × 10 × 5.7 = SPAG9 Supplementary Data Set 7 Set Data Supplementary g * + – ) RT–PCR analysis of alternatively spliced cassette exons in exons cassette spliced alternatively of analysis ) RT–PCR HFFs 276 during HCMV and mock infection of HFFs ( HFFs of infection mock and HCMV during ′ ± 2 -end-processing-factor control. Uncropped blots are are blots Uncropped control. -end-processing-factor UTR 200 bp 2 −17 SYNRG d and and *** , we reasoned that CPEB1 might also modulate the the modulate also might CPEB1 that reasoned we , HFF NPC ) Global analysis of the presence of both a canonical a canonical both of presence the of analysis ) Global * ± n 23 × 10 × 2.36 and HCM proximal

−7 USPL1 −18 nature structural & molecular biology molecular & structural nature ± U 250 bp Distal V 369 after lentivirus-mediated CPEB1 OE (compared (compared OE CPEB1 lentivirus-mediated after ) in HCMV-infected HFFs. These categories categories These HFFs. HCMV-infected in ) *** b 125 119 and 1.5 × 10 × 1.5 and ). We concluded that CPEB1 KD reverses reverses KD CPEB1 that concluded We ). SYNRG c - - | ) qRT–PCR analysis of distal 3 distal of analysis ) qRT–PCR mRNA transcripts after OE of GFP (control), (control), GFP of OE after transcripts mRNA ′ During infection: CPE -UTR shortening. -UTR g Not changed Extended Shortened a n ) Immunoblot of lentivirus-mediated lentivirus-mediated of ) Immunoblot = 1 experiment per condition per per condition per = 1 experiment c | PAS and and Control . . ( f P −9 ) Venn diagram showing overlap of overlap showing ) Venn diagram = × 7.61 10 b 2 kb ) and CPEB1 OE (shortening (shortening OE CPEB1 and ) −8 ) showed an enrichment in the the in enrichment an showed ) ) RNA-seq coverage of the 3 the of coverage ) RNA-seq d

′ CPEB1 ) altered events ( events altered ) -proximal termination sites in sites termination -proximal are available online. available are ′ -UTR shortening events, events, shortening -UTR HCMV PAS Mock e APA CSTF-77 CPEB1 OE | P (427) < 0.0001; * < 0.0001; −14 ±

M c s.e.m. ( s.e.m. Usage (distal/total) ) ) and appa ‘Golgi 0.0 0.2 0.4 0.6 0.8 1.0 1.2

V-48 128 Mock n ′ -UTR usage or usage -UTR = 1 RNA-seq = 1 RNA-seq n

Fig. 5 Fig. HCMV HCM = 3 qRT– n V-96 (473) SYNRG = 1 P < 0.01. < 0.01.

USPL1 SPAG9 MYO18A GFP V

d CPEB1 ′

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© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. (+) HFFs showed a decrease in median poly(A)-tail lengths after after lengths poly(A)-tail median in decrease a showed HFFs (+) siCPEB1 and (+) infected in transcripts HCMV of TAIL-seqanalysis CPEB lengths. including poly(A)-tail host changes, RNA-processing infection-induced of fraction cluded that CPEB1 depletion during HCMV infection reverses a large ( ( ment enrich showed depletion CPEB1 after tails poly(A) shortened had ( infection mock after observed levels the to lengths tail median the decreased ( infection HCMV during that the median poly(A)-tail lengths of host transcripts were with increased analysis length TAIL-seq poly(A)-tail transcriptome-wide performed We genes. viral and host both of transcripts of lengths poly(A)-tail MYO18A in (boxed) exons AS of samples; OE GFP and siCPEB1, HCMV-infected siRNA, NT HCMV-infected siRNA, NT ∆ in reported as reversal, showed exons 472 samples. across a comparison in values, splicing-index mean the to relative values) (negative exclusion and values) (positive inclusion exon of scale the shows bar color The 3). (MOI AS HCMV-infection-related of ( 4 Figure nature structural & molecular biology molecular & structural nature ( depletion CPEB1 a Supplementary Data Set 8 Set Data Supplementary I ) Hierarchical clustering of alternatively spliced exon indices (as a measure of exon inclusion by RNA-seq in HFFs). Boxed regions highlight a reversal a reversal highlight regions Boxed HFFs). in RNA-seq by inclusion exon of a measure (as indices exon spliced alternatively of clustering ) Hierarchical (a measure of change in exon inclusion) >|0.1|). HCMV-infected cells are indicated by +. For RNA-seq, RNA-seq, For +. by indicated are cells HCMV-infected >|0.1|). inclusion) exon in change of measure (a 1 P

depletion , , = 6 × 10 × 6 = Genome-wide host AS remodeling in HCMV infection is replicated by CPEB1 overexpression and corrected by CPEB1 depletion. depletion. by CPEB1 corrected and overexpression by CPEB1 is replicated infection in HCMV AS remodeling host Genome-wide SPAG9 2 5 a Fig. 5e Fig. ( Fig. 5 Fig.

, , Mock TTC7A

, −21 shortens f e ). GO analysis of host genes whose transcripts transcripts whose genes host of analysis GO ). i. 6 Fig. and and ITGA6 ) in the ‘membrane-enclosed lumen’ category category lumen’ ‘membrane-enclosed the in ) GFP OE and and Supplementary Fig. 6a Fig. Supplementary a and and

). For example, the HCMV transcript transcript HCMV the example, For ). ITGA6 poly(A) i. 5 Fig.

and and siCPEB1/+ SPAG9 Supplementary Fig. 6b Fig. Supplementary in either mock (−)- or HCMV (+)-infected cells treated with either NT or CPEB1 siRNA. CPEB1 or NT either with treated cells (+)-infected HCMV or (−)- mock either in

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transcripts AS index AS 0.36 0.01 0.04 b –0.51 –0.34 –0.17 ). We con ). 178 185 212 384 0.00 0.17 0.34 0.51 63 1 1 ______5 ′ + =HCMV c - - ) RT–PCR analysis of AS cassette exons (4/4 selected and validated) of validated) and selected (4/4 exons cassette AS of analysis ) RT–PCR

alterations in length, we poly(A)-tail immunoblot performed analysis accompanied also levels protein viral in changes whether assess To ( detection length poly(A)-tail for reads UL55 increased three-fold and five-fold over the control, respectivelyprotein ( ( mental-retardation X fragile RBP unrelated the of binding to relative length ( transcript, which had statistically significant differences in poly(A)-tail Indeed, we observed highly increased CPEB1 binding to the the to binding CPEB1 increased highly observed we Indeed, transcripts. viral to binds directly CPEB1 whether evaluate to PCR) cross-linking and immunoprecipitation coupled with qRT–PCR (CLIP–cant change (Mann–Whitney decreased after CPEB1 depletion, whereas the in length Poly(A)-tail ( length poly(A)-tail median system immune host UL18 c ITGA6 MYO18A SPAG9 TTC7A HCMV ITGA6 48 hpi , an MHC I class homolog that participates in evasion of the the of evasion in participates that homolog class I MHC an , on b CE1 a nt upre b sfiin TAIL-seq sufficient by supported not was CPEB1 by bound Fig. 6 n Fig. 6 = 2 experiments for CPEB1 OE samples. ( samples. OE CPEB1 for = 2 experiments

b + – c and ). CPEB1 binding to NT Supplementary Data Set 8 3 3 5 ′ , showed the largest decrease in mean and and mean in decrease largest the showed , 179 281 212 305 183 – n UL83 siCPEB1 = 1 experiment each for mock-infected mock-infected for each = 1 experiment 1 1 1 1 1 Supplementary Data Set 6 Set Data Supplementary Fig. Fig. 6 ______5 ′ U + test; – UL82 b UL83 and SPAG P = 0.06; bicistronic transcript was also also was transcript bicistronic and Supplementary Data Set 8 Set Data Supplementary Supplementary Data Set 8 Data Set Supplementary 9 UL54 UL55 ) after CPEB1 depletion, Fig. Fig. 6 did not show a signifi b ) RNA-seq coverage coverage ) RNA-seq CPEB1 OE GFP siCPEB1/+ NT/+ Mock transcripts was also 3 s e l c i t r a ′ b (FDR <0.05, <0.05, (FDR ). ). We performed

Fig. 6

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© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. n iPB HF cmae wt N-iN-rnfce HF at ( HFFs h 96 NT-siRNA-transfected with compared HFFs siCPEB1 in decrease ten-fold approximately an showed assay plaque-forming a of healthythose mock-infected cells. to Productive HCMV titers establishedsimilar with characteristics fibroblast-like maintained HFFs infected in effects ( siRNA control NT with treated the cells with compared as infection, after observed normally effects of cytopathic the rescue visible a dramatic exhibited hpi 48 at depletion CPEB1 with 0.5) (MOI HFFs infected the of morphol ogy The HFFs. HCMV-infected siCPEB1, infec analyzed we viral on tion, depletion CPEB1 of effects cellular the evaluate To CPEB teins and IE-72 showedIE-86 no changes ( depleted HCMV-infected (+) HFFs, whereas the immediate early pro proteinviral pp28 late (encoded bythe of level the that revealed results Our proteins. viral of s e l c i t r a  the of Notably, overexpression for TOE1 and RAB5C, respectively: M, M, respectively: RAB5C, and TOE1 for (si) siCPEB1 versus infected for lengths poly(A)-tail of distribution ( (+HCMV). HFFs siCPEB1 and controls mock-infected versus 3) (MOI HFFs HCMV-infected in analysis) final for pooled were runs ( Cor, OE. correction. CPEB1 versus OE GFP and (+HCMV); (si) siCPEB1 versus HCMV (V); HCMV versus (M) mock in category exosome extracellular the in enrichment progressive showing analysis, ( replicates in the of coverage ( sets data RNA-seq two between common >10,000) factor (Bayes transcripts 196 for cells ( 5 Figure a

) Heat map showing 3 showing map ) Heat SYNRG 1 Fig. 7 Fig.

is HCMV-infection-related host genome-wide 3 genome-wide host HCMV-infection-related and and

necessary n d a = 3 qRT–PCR reactions). ** reactions). = 3 qRT–PCR –log P (FDR cor.) b 10 ANKH ). The titers recovered, at least partially, by 144 hpi. hpi. 144 by partially, least at recovered, titers The ). 16 12 SYNRG 0 4 8 Mock vs. M vs. HCMV GO: extracellular transcripts in either mock (−)- or HCMV (+)-infected cells treated with either NT or CPEB1 siRNA (si-17). Error bars, s.d. among among s.d. bars, Error (si-17). siRNA CPEB1 or NT either with treated cells (+)-infected HCMV or (−)- mock either in transcripts

V for exosome 3

′ GFP vs. CPEB1V vs. si -UTR shortening (yellow) and lengthening (blue) in mock-infected versus HCMV-infected and HCMV-infected versus siCPEB1 siCPEB1 versus HCMV-infected and HCMV-infected versus mock-infected in (blue) lengthening and (yellow) shortening -UTR HCMV vs. ′ UTR under all conditions in HFF cells. HCMV-infected cells are indicated by +. ( +. by indicated are cells HCMV-infected cells. HFF in conditions all under UTR siCPEB1(+) productive UL99 P < 0.0015 and and < 0.0015 APA ) was consistently lower in the CPEB1- UL99 Short Long –0.90 –0.60 –0.30 0.00 0.30 0.60 0.90 TOE1 n e

-encoded protein pp28 alone in in alone pp28 protein -encoded HCMV = 22, 28; V, 28; = 22, Fig. 7 Fig.

P Probability density < 0.005; * < 0.005; 0.06 0.02 0.04 and and 0.0 e b ) Median poly(A)-tail lengths determined by TAIL-seq ( TAIL-seq by determined lengths poly(A)-tail ) Median Fig. Fig. 6 P 2 0 a < 2 × 10

). The infected siCPEB1 siCPEB1 infected The ). RAB5C infection = 20reads minimum coverage HFF hostgenes 0.01 0.01 0.03 0.01 222 127 103 171 TAIL -seq 72 1 d ______). ′ n P -UTR shortening and poly(A)-tail lengthening in HCMV infection is reversed by CPEB1 depletion. depletion. CPEB1 by reversed is infection HCMV in lengthening poly(A)-tail and shortening -UTR 4 0 = 25, 43; si, si, 43; = 25, < 0.05 by one tailed Student’s Student’s tailed one by < 0.05 transcripts (mock-infected (M) versus infected (V) (V) infected versus (M) (mock-infected transcripts −6 by one-sided Mann–Whitney Mann–Whitney one-sided by 6 0 advance online publication online advance CPEB1 OE siCPEB1/+ 8 0 n SYNRG Mock NT/+ = 33, 63. Source data for for data Source 63. = 33, GFP - - - 0 (8,196 genes) siCPEB1 48hpi (7,956 genes) HCMV 48hpi (8,131 genes) Mock 48 As an orthogonal approach to deplete CPEB1, we used uniformly uniformly used modified 2 we CPEB1, deplete to approach orthogonal an As ( depletion CPEB1 after decreased also was pp65, encoded in cells ( CPEB1-specific siRNA recapitulated the infection-related morphology the to insensitive was that fusion CPEB1-GFP codon-optimized a of related morphology at 48 hpi,infection- whereas lentivirus-mediated in expression result not did cells HCMV-infected siCPEB1-treated, Human gene transcripts are subject to post-transcriptional post-transcriptional to during length subject poly(A)-tail and APA AS, of are level the at regulation transcripts gene Human DISCUSSION ( infection productive supporting and genes HCMV and of host RNA the landscapes for shaping is necessary CPEB1 that changes in AS the and rescued levels protein pp28 viral the decreased of CPEB1 9 Set Data phosphorothioate backbone against exon 5 in 100 h Supplementary Fig. 6c Supplementary t test with unequal variances. ( variances. unequal with test 120 U ′ and and SPAG9 test, respectively). Numbers of transcripts ( transcripts of Numbers respectively). test, - c O- n

= 1 RNA-seq experiment per condition). ( condition). per experiment = 1 RNA-seq Usage (distal/proximal) methoxyethyl antisense oligonucleotides (ASOs) with a f Supplementary Fig. 6d Fig. Supplementary 0.6 1.0 Tail length (nt) 0.2 ( c 150 200 250 100 –

50 Supplementary Fig. 6f Supplementary 0 e n nature structural & molecular biology molecular & structural nature are available online. available are = 2 MiSeq runs per condition, data sets from two two from sets data condition, per runs = 2 MiSeq NT/– TOE1 V M ** c P SYNRG ) qRT–PCR analysis of distal 3 distal of analysis ) qRT–PCR < 0.0025 and and < 0.0025 NT/+ * ). ). Indeed, another late protein, si d ) DAVID gene ontology (GO) (GO) ontology ) DAVID gene , si-17/– e RAB5C ). ASO-mediated depletion depletion ASO-mediated ). P ). ). Therefore, we concluded V M < 5 × 10 CPEB1 ** ANKH f ) Violin plots showing showing plots ) Violin * si-17/+ ( n b −5 Fig. 7 Fig. Supplementary ) are as follows follows as ) are ) RNA-seq ) RNA-seq si , respectively; , respectively; ′ -UTR usage usage -UTR d ). Fig. 7 Fig. UL83 c ). ). -

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. which which can change of the cellular environment in either support of decay, viral replica nonsense-mediated and frameshifts through levels protein decreasing by or transcripts of sequence coding the altering by either proteins of different production to the lead can AS changes HCMV. by affected pathways cell-type-independent shared, gesting measurements Transcriptome-wide commonly revealed altered AS events in different cell thus types. types, sug cell infected among processing different RNA of level the at convergence a observed HCMV, we distinguishes that tropism cellular wide the despite infec study, viral by our In unclear. remains affected mechanisms, underlying are the as well as tion, transcripts host in events processing cancer and diseases cular development (mixture) of four different siRNAs against CPEB1. Uncropped blots are shown in siRNA (siNT) or four different CPEB1-targeting siRNAs (si-02, si-04, si-17 and si-18) at MOIs of 0.5 and 3. ( transcripts ( for RNA targets bound by CPEB1 and FMRP. IP, immunoprecipitation. Error bars, s.e.m. among replicates ( containing and HFFs and infected siCPEB1 HFFs. ( ( 6 Figure nature structural & molecular biology molecular & structural nature favored of the majority transcripts the altered infection, During tion. d n ) Immunoblot analysis of CPEB1 and viral proteins (IEs, UL57 and pp28) in mock (−)- or HCMV (TB40E; +)-infected cells treated with either nontargeting = 2 MiSeq runs per condition, data sets from two runs were pooled for final analysis) of median poly(A)-tail lengths of viral transcripts in HCMV-infected

CPEB1 depletion by siRNA results in shortened poly(A)-tail lengths and decreased protein levels for HCMV late genes. ( n c b a

) are as follows for UL18, UL54, UL99 and UL83-82, respectively: HCMV, Fraction Frequency 2.7 17 0.02 0.04 0.06 UL54 1. 0. 0. 0. 0. 0. 1. 0. 0. 0. 0. 0. 0.

, Fold increase over FMRP 18 0 2 4 6 8 0 0 2 4 6 8 0 × 0 10

IP (normalized to mock) 20 ,

24 0 UL99 UL18 0 2 4 6 8 5 HCMV transcripts ( HCMV genes , 3

6 UL44 50 . AS and APA are also perturbed in neuromus in . APAand AS perturbed are also Median poly(A)-taillength(nt) P 40

UL44-2 =1.06 100 P CPEB1 KD HCMV CPEB1 IP FMRP IP =0.00001 CPEB1 KD HCMV 17 , × 19 60

150 = 20reads Minimum coverage UL54 10 Tail length(nt) , 3 –5 7 b (74 genes) siCPEB1 48hpi (77 genes) HCMV 48hpi . However, the scale at which RNA which at scale the However, . 200 ) Cumulative distribution frequency (CDF) plots of poly(A)-tail length distributions for

UL55 80 0 P UL54 UL83-82 < 10

50 100 IE P =1.26

−5 UL99 100 P CPEB1 KD HCMV CPEB1 KD HCMV , =0.07 120 P × 10 < 0.00014, UL83 150 –21 advance online publication online advance 200 P d < 10 hp 96 48 hpi 96 hpi 48 hpi i −20 + – - - - - and SP processing changes during virus infections will provide key insights insights key provide will infections virus during changes processing proliferation. of effect tive, thus that indicating 3 global prolifera not are cells the infection, HCMV itself lytic during infection However, the of than rather proliferation associated consequence the a of be to appear patterns polyadenylation host altered the and cells, B immortalizes and infection latent a establishes virus transcripts host of number a for lengths B cells infected with Epstein–Barr virus demonstrate decreased 3 cancer as such states proliferative highly in occur translation of 3 the transcripts. affected that UTR may lengths to lead changes and/or in stability, 3 localization decreased in resulting thereby site, poly(A) proximal a of usage Supplementary Data Set 10 P < 0.06, by one-sided Mann–Whitney – Identification of RBPs that are responsible for host and viral RNA-of for and RBPs host viral that are responsible Identification

NT + n = 91, 93, 1,022, 1,034; CPEB1 KD, – + –

si-02

si-04 + – n = 3 qRT–PCR reactions). Numbers of . Source data for β NT -actin is a loading control. SP, Smartpool – + ′ -UTR shortening -UTR is shortening not a secondary si-17 + U test, respectively). ( – MOI =0.5 MOI = UL18 ′ -UTR -UTR shortening is known to si-18 a + 3 – 9 c n 3 . However, Epstein–Barr Epstein–Barr However, . , a are available online. = 78, 54, 1,341, 1,207. UL99 ) TAIL-seq distributions CPEB1 Acti pp28 UL5 IE1 72 pp28 Acti IE1 72 IE2 86 IE2 86 CPEB1 n n 7 37 , s e l c i t r a UL83-82- , 3 8 . Furthermore, Furthermore, . c ) CLIP–PCR ′ -UTR 3 ′ 9  - - .

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. splicing factor U2AF65 (ref. (ref. U2AF65 factor splicing function neuronal and development early in role essential an plays that RBP well-studied a is CPEB1 analyzed. types cell primary three all in CPEB1 RBP of the are upregulation and consistent Weobserved transmission targets. therapeutic and potential propagation viral of mechanisms into s e l c i t r a  (ref. CSTF-64 and CPSF-73 HCMV titers. Source data for for data Source titers. HCMV 3 normal of preservation to leads depletion CPEB1 contrast, In titers. HCMV 3 shorter isoforms, AS certain supports turn in which expression, CPEB1 induces infection HCMV CPEB1. of (right) absence and (left) presence the in ( in shown are blots Uncropped control. is a loading GAPDH (si17). siRNA NT or CPEB1-targeting with treated or were untreated left were +) that 0.5; (MOI cells (TB40E)-infected HCMV and (−)- in mock UL83) and CH160 (IEs proteins viral and of CPEB1 ( units. plaque-forming Pfu, replicates. among s.d. (siCPEB1). siRNA CPEB1 with treated cells TB40E-infected and (siNT) NT with treated cells TB40E-infected TB40E), (HCMV cells TB40E-infected for assay a plaque by determined as points, time different at titers viral ( shown. are images representative and times, three repeated was experiment The magnifications. 10×) (bottom, high and 4×) (top, low at (siCPEB1) siRNA CPEB1 or NT either with treated cells ( infection. 7 Figure d

b d a (rounded morphology HCMV infecte HFF ) Working model summarizing our findings regarding HCMV infection infection HCMV regarding findings our summarizing model ) Working

Long poly(A)tail Titer (pfu/ml) HFF HFF

10 10 10 10 10 4

10 × × 2 3 4 5

′ ′ AAAAAAAAAAAA CPEB1 depletion rescues cytopathology and attenuates HCMV HCMV attenuates and cytopathology rescues depletion CPEB1 -UTR lengths and poly(A)-tail lengths, and decreased productive productive decreased and lengths, poly(A)-tail and lengths -UTR UTRs, longer poly(A)-tail lengths and normal productive productive normal and lengths poly(A)-tail longer UTRs, HCM virion 48 a n ) Phase-contrast images of mock- or HCMV (TB40E)-infected (TB40E)-infected HCMV or mock- of images ) Phase-contrast siCPEB1 siNT HCMV TB40 siNT d Mock NT = 3 independent plaque measurement assays. Error bars, bars, Error assays. measurement plaque = 3 independent V 72 A Nucleus ) APA 1,000 200 Time (hpi) E µ µ m m 96 AS 22 CPEB1 , 32 b 120 , 4 are available online. available are 4 0 3 1 . CPEB1 is known to interact with the the with interact to known is CPEB1 . 2 ). CPEB1 binds a consensus sequence sequence consensus a binds CPEB1 ). TB40E NT ) and the 3 the and ) 144 morphology Flattened fibroblast-like Supplementary Data Set 10 Set Data Supplementary

Short poly(A) tail 1 ,000 AAAAA c 200 µ AS µ m – m ′ -end-machinery proteins proteins -end-machinery HCMV infected HFF CPEB1KD, APA c + + + ) Immunoblot analysis analysis ) Immunoblot b ) Productive HCMV HCMV ) Productive TB40E siCPEB1 siCPEB1 siNT Viral proteins CPEB1 RNAi knockdown siCPEB1 1 ,000 200 advance online publication online advance GAPDH UL83 CH160 CPEB1 HCMV . µ µ

m m

MOI = 0.5 cleavage sites compared with unaffected 3 unaffected with compared sites cleavage KD cells KD CPEB1 in efficiency translational decreased a and tail poly(A) short senescence polyadenylation and translation of gene transcripts involved in cellular host infection, rejection allograft and evasion viral system immune of progression packaging, viral to contribute can terms) GO (enriched microvesicles and exosomes Furthermore, contains markers for both the because HCMV packaging occurs in a salient membrane-enclosed compartment that particularly was finding This apparatus. Golgi and exosome extracellular of categories the in enrichment showed tion) infection, CPEB1 overexpression and CPEB1 depletion (during infec APA in during altered sets Gene infection. mock after observed that hundreds of RNA processing events and reversed produced a pattern similar to infection HCMV during depletion CPEB1 Similarly, tion. APA events reminiscent of RNA-processing patterns in HCMV infec and AS of fraction substantial a to led cells noninfected in CPEB1 of 3 HCMV-infection-altered near CPEs in enrichment striking reporters minigene and transcripts date signals poly(A) to 3 and introns in CPE, the termed (UUUUUAU), CPEB1 depletion was not sufficient to generate HCMV cytopathology. of expression the in decrease a to delivered transiently led KD of CPEB1 infection, During CPEB1. ofresurgence a siRNAand loss eventual of because probably points, matelyhpi.titerseventuallyrecoveredten-foldat 96 The at later time approxiby titers HCMV productive the decreased also but changes cytopathic these reversed only not CPEB1 of depletion that strated (refs. hpi 48–72 at inclusions nuclear and nuclei assembly. viral in involved nents exosomes and Golgi host with also and other each with interact respectively) UL83 phosphoprotein that inhibits cellular antiviral response. Notably, both pro is and attenuated duce infection virions, not do knockouts pp28 that shown has envelopment, report previous viral a and final for essential is that protein tegument a is containing RNAs viral with knockdown during infection. Furthermore, CPEB1 directly interacted CPEB1 after expression protein decreased and tails poly(A) shorter HCMV assembly HCMV in important is which RAB27A, including altered, were GTPases) small and RABs (including family poly(A)-tail lengths of many members of the small Ras-related protein protein thus localization, suggesting viral assembly pathways. Indeed, and lumen membrane-enclosed of categories the in enrichment an scripts had shorter poly(A)-tail lengths after CPEB1 depletion showed the GO of tail lengths analysis transcripts. viral host tran genes whose shortened the decreased only also but levels not normal to lengths infection poly(A)-tail median HCMV during CPEB1 of tion deple siRNA-mediated Furthermore, tails. poly(A) lengthened with of gene Wethe host transcripts expression decreased did not observe nuclear retention and gene decreased expression of host transcripts extension, protein SOX in the viral tion, which in results poly(A)-tail contrast with Kaposi’s sarcoma–associated (KSHV) herpesvirus infec in was phenomenon This infection. HCMV during transcripts host of lengths poly(A)-tail in increase an showed TAIL-seq with lengths In the cytoplasm, CPEB1 is linked to the alteration of cytoplasmic of cytoplasmic alteration the to linked is CPEB1 cytoplasm, In the After HCMV infection, cells became rounded and had bulging bulging had and rounded became cells infection, HCMV After and and UL99 5 0 55– . In our study, transcriptome-wide analysis of poly(A)-tail of poly(A)-tail analysis study,. In our transcriptome-wide UL99 50 5 , 5 7 and and 1 . Therefore, CPEB1 affects both host and viral compo and host viral both affects . CPEB1 Therefore, UL99 . For example, p53 ( p53 example, For . gene products (tegument proteins pp65 and pp28, pp28, and pp65 proteins (tegument products gene 42– UL83

ln b uig etvrss n h cnet of context the in lentiviruses using by alone UL99 nature structural & molecular biology molecular & structural nature 4 4 and further affects AS affects further and contained in multicistronic transcripts had had transcripts multicistronic in contained product pp28. We found that exogenous that found We pp28. product UL99 trans -Golgi network -Golgi and endosomes and and TP53 5 UL83 ) mRNA has an abnormally abnormally an has mRNA ) 4 3 . . 2 ′ UL83 -ends. Ectopic expression expression Ectopic -ends. . Our analysis revealed a revealed analysis Our . . . 4 46– UL99 1 encodes a tegument encodes and APA in candi in APA and 58 4 5 9 ′ , 3 UTRs proximal proximal UTRs . 5 . We found that that Wefound . -encoded pp28 pp28 -encoded 9 ). Wedemon ). ′ -end -end 4 5 5 2 ------. .

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. Note: Any Supplementary Information and Source Data files are available in the number accession under database Omnibus Expression Gene the in deposited been have data codes. Accession the in v available are references associated any and Methods M RBP-RNA host the infections. DNA in virus uncover dysregulated to networks needed be will efforts further and family, may to mechanisms host extend of other members the herpesvirus HCMV.These for target therapeutic potential a infection provides and HCMV to susceptibility cellular of mechanisms on light sheds study this Finally, landscapes. RNA viral and host affect that minigene reporters and to understand the roles of other cellular RBPs and binding CPEB1 RNA between with processing connection direct to a of establish mechanisms induction, CPEB1 to important identify be will work Future involved. be may RBPs infections, other that likely is it viral and other or HCMV establish to landscape RNA host the shapes that factor host only the be not may CPEB1 interactions. nylation in host-virus interactions and identifies a new player in these polyade alternative host modulating of importance the of evidence tion in productive HCMV in infection HFFs. Our study provides new and inhibition of late infection stages are in strong agreement. (later stages in infection), our two results of viral packaging dysfunction alters host and viral transcripts involved in viral packaging but andCPEB1 is transportrequired for late stages of virus infection. Because CPEB1 72 were not affected, thus suggesting that viral infection is established, IE and 86 IE proteins early immediate The infection. HCMV during late the Indeed, infection. virus in important proteinsbe mayand scripts This result suggests that CPEB1-mediated effects on other HCMV tran nature structural & molecular biology molecular & structural nature assays. F.R. provided the antisense oligonucleotides. microarray analysis. R.B., T.J.S. and B.T.R. performed RT C.G.-B. performed microarray hybridizations. S.C.H., J.P.D. and M.A. performed APA assays. H.H. and R.B. prepared TAIL-seq libraries, as overseen by S.A. bioinformatics analysis. R.B. and T.J.S. performed RT–PCR splicing and E.C.W. made RNA-seq libraries. R.B., T.J.S., S.C.H., B.A.Y. and S.S. performed and microscopy. T.J.S. and T.J.B. made lentivirus preparations. R.B., T.J.S. and T.J.S., E.C. and R.B. performed western blots. R.B. performed immunofluorescence HCMV infections; R.B., T.J.S. and E.C. performed siRNA treatments. E.C. and J.-P.B. maintained HCMV viral stocks, measured titers and performed R.B., T.J.S., D.H.S. and G.W.Y. the designed study and wrote the manuscript; postdoctoral fellow. G.W.Y. is supported as an Alfred P. SloanGraduate Research ResearchFellow. Fellow. R.B. is supported as a Myotonic Dystrophy Foundation Institute of General Medical Sciences, T32 GM008666. E.C.W. is supported as an NSF Genetics Training Program through an institutional training grant from the National T.J.S. and E.C.W. are supported in part by the University of California,California San InstituteDiego, of Regenerative Medicine (RB3-05219 to G.W.Y. and D.H.S.).Institutes of Health (HG004659, HG007005 and NS075449 to G.W.Y.) and frominfected the materials. This work was partially supported by grants from the National with HSV-2 infections, and the laboratory of S.A. Spector (UCSD) for HIV-1- protocol before publication. We especially thank C.S. Morello (UCSD) for assistance S. Azoubel Lima and A. Pasquinelli (UCSD) for sharing their modified TAIL-seq N. Kim, Seoul National University) for help with the Tailseeker algorithm, and (UCSD) for initial assistance with bioinformatics. We thank H. Chang (laboratory of reading of the manuscript and extend particular acknowledgment to M.T. Lovci The authors thank members of the laboratories of G.W.Y. and D.H.S. for critical v AUTH A e e c r ethods r In summary, we demonstrated an roleunexpected of CPEB1 induc s kno s i o UL83 i n o

O n o f w R R C

o t h f product pp65 was also downregulated after CPEB1 depletion l e

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e p r . a p Sequencing data and splicing-sensitive microarray microarray data and splicing-sensitive Sequencing n e r ts . ON S G S E 7 4 2 5 0 . –

PCR PCR splicing and APA advance online publication online advance

o

n o n l i l i n n e - - - e

10. 9. 8. 7. 6. 5. 4. 3. 2. 1. r at online available is information permissions and Reprints v The authors declare competing interests:financial details are available in the 30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. C e e O p

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MPETI Sydiskis, R.J. & Roizman, B. Polysomes and protein synthesis in cells infected with infected cells in synthesis protein and Polysomes B. Roizman, & R.J. Sydiskis, Fenwick, M.L. & Walker, M.J. Suppression of the synthesis of cellular macromolecules R. Horváth, for target novel a as Cytomegalovirus R. Khanna, & D.G. Walker, A., Schuessler, endothelial vascular of Infection D.H. Spector, & S. Chien, J., Li, development. J.B., DuRose, atherosclerosis and Infection M.E. Rosenfeld, & L.A. Campbell, in Cytomegaloviruses. R.F. Pass, & T. Shenk, E.S., Mocarski, cellular of Exploitation Spector,D.H. & I. Sanchez, McElroy,A.K., E.A., Fortunato, Mocarski, E.S. Betaherpes viral genes and their functions. in Staras, S.A. Belzile, J.P., Stark, T.J., Yeo, G.W. & Spector, D.H. Human cytomegalovirus infection L.T. Gehman, of design and Analysis S.C. Huelga, C.B. Burge, & E.M. Airoldi, E.T., Wang, Y., Katz, D.D. Licatalosi, of determination genome-wide TAIL-seq:V.N. Kim, & M. Ha, J., Lim, H., Chang, Poly(A)-tail D.P. Bartel, & H. Sive, G.R., Chen, S.W., Eichhorn, A.O., Subtelny, 3 and (PAP) polymerase Polyadenylate A. Scorilas, Richter,J.D.translation.lifeCPEB:ina E. Klerk, de M. Jenal, K. Charizanis, disease. in mis-splicing RNA M.S. Swanson, & M.M. Scotti, R. Batra, in Developments Ulitsky,I. M.S. Swanson, & K. Charizanis, R., Batra, M.G., Poulos, N. Stern-Ginossar, Gatherer, D. during late expression gene cell host of analysis Global E.S. Mocarski, & L. Hertel, function(s) a contain cells virus-infected simplex Herpes N. Frenkel, & A.D. Kwong, by herpes simplex virus. simplex herpes by atherosclerosis. of multiforme. glioblastoma of immunotherapy Virol. J. artery. the of regions atheroprone simulating conditions flow in virus of spread and entry preferential reveals stress shear fluid under cytomegalovirus human with cells Res. Med. (Lippincott Arch. 2701–2772 P.M.) Howley, & D.M. Knipe, 2007). (eds. Wilkins, & Williams 2 Vol. edn, 5th (2000). 111–119 cytomegalovirus. human by pathways regulatory and signaling 2007). Press, University Immunoprophylaxis (Cambridge and Therapy, Biology, 1988–1994. (2014). DNA. viral of persistence the to leads but at steps several restricted is cells stem neural primitive cell-derived stem embryonic human of brain. mammalian the in excitation proteins. hnRNP by splicing alternative of (2010). 1009–1015 regulation. isoform identifying for experiments sequencing RNA processing. RNA 3 and length tail poly(A) (2014). control. translational in switch embryonic an reveals profiling disease. (2002). with 193–224 association and assays, function, polyadenylation. sites. polyadenylation and cleavage (2012). dystrophy. myotonic in dysregulation and brain developing (2016). 19–32 disease. (2014). RNA-mediated in polyadenylation alternative Res. Genome disease. (2011). and splicing RNA (2012). 1088–1093 USA Sci. Acad. gene cycle cell 78 of dysregulation function. US28 of extensive independent pseudomitosis of induction and expression reveals infection cytomegalovirus mRNAs. viral (1987). 1926–1930 and host both destabilizes that virus. DNA a i i n o t n , 11988–12011 (2004). 11988–12011 , s

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© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. mination sites with the highest coverage were selected to define (withputative filtering proximalperformed for genomic regions with A tracts). Finally, the two teringreads were on human-mappedbased poly(A) threshold of five reads required to constitute a poly(A) site. Thesethanone cleavage site detectedby RNA-seq cleavage-definwere considered, minimumwitha 3 define (to v2.16.2 Bedtools e tations, we downloaded all Ensembl-defined human 3 with the high Bayes factor for the HCMV samples). To generatedegrees of UTR shift from this value upward,the when itcustom was used in combination anno 0.03 (although the cutoff selected for this latter value thresholdwas of low,10,000 and difference valueswe ( found significant exceptcustomoffor3 use analysis was performed with the MISO algorithm v0.5.2 with default settings previouslysoftwaresuiteasQuantasdescribed the cycles of PCR amplification. For AS analysis of RNA-seq data, we (reverseused transcriptionOlego andperformed with Superscript III, Life Technologies) and 35 assays were performed with the equivalent of 50 ng of oligo(dT)-primed cDNA microarray analysis was performed as previously described Analysis of human alternative splicing and polyadenylation. and Stern-Ginossar Merlin reference to facilitate comparisons to previous studies by Gatherer HCMV ORF annotations. Data processing was also performed with the HCMV K (GenBank sequence genome TB40E available newly the used we urements, expression. gene HCMV of Analysis HSV-2 samples were multiplexed and run together. preparedIlluminawithTruSeq StrandedmRNA Sample Preparation reagents. were infections HSV-2 of analysis for Libraries results. clustering expression gene- hierarchical from results visualize and perform combinationto in used were TreeviewJava and 3.0 Cluster together. run and multiplexed barcoded, samplewas platform. Each HiSeq Illumina an on sequencedsamples were All reagents.Preparation Sample mRNA poly(A) TruSeq Illumina with prepared comparativeHCMV,mock, analysisof sampleswereOE CPEB1 and siCPEB1 Primer,IntegratedPrimers,ReadyMade DNA Technologies). the forLibraries ing oligo(dT) was included during first-strand cDNA synthesis (cDNA Cloning indetail specific libraries with the dUTP method mock-infectionconditions,TB40E-infectionandHCMVprepared westrand- TRIzol reagent, per the manufacturer’s protocol (Life Technologies). For all the RNA-seq library preparation and data processing. 5 d after infection. performed as described snap frozen before subsequent analysis. HIV-1 infections of T-cell cultures were and pelleted briefly trypsinization, by harvest of time the at collected were als HSV-2hpi.andHCMV8 orAll at2 harvestedandmateri °Cincubated at37 initial 30-min adsorption period at 4 °C, mock-infected and infected cells were tions were performed in HFFs of strain G at an MOI of 10. For HSV-2, after an 400 mJ/cm of dose a to exposed was HCMV treatment, UV For HFFs. of treatment the IFN- recombinant IFN- For 3. of MOI an at conducted were 3d Fig. ( comparisonsfollow-up extended in used were cells derived described infections. Virus for mycoplasma. negative found were and (Lonza) kit test mycoplasmaMycoAlert a with tion 3 humanWecells.ES humanprimary foreskin used fibroblastsref. in as (HFFs) types. Cell ONLINE doi: and distal alternative ends of the 3 n 0 F s , and ECs as ref. as ECs and , 2 e 10.1038/nsmb.3310 9 m 7 b 3 , 3 l e 2 . 9 ). Towne). HCMVinfections(RT–PCR comparisons shownin o 8 3 .For the TB40E and mock-infected HFFs and ECs, adaptor-contain . r 1 0

2 g Neural precursor cells (NPCs) were derived from H9 and HUES9 and H9 from derived were (NPCs) cells precursorNeural METHODS . HUES9-derived NPCs were primarily used in this study,this HUES9-derivedwerein primarilyNPCs. used andH9- , strain ‘Lisa’) for mapping and analysis of coverage of GenBank-listed of UV light in a Stratalinker cross-linker (Stratagene). HSV-2 infec / , release 75) and flattened them with the mergeBed function from All TB40E HCMV infections were performed as previouslyas performed wereinfections HCMV TB40E All γ 6 et al. Acm b69 i MM ls 0 FS a ue for used was FBS 10% plus MEM in ab9659) (Abcam . Cells were routinely tested for mycoplasmacontaminafor testedroutinely were Cells . 6 0 13 and harvested directly into TRIzol (Life Technologies) ′ , 1 -UTR-isoformannotations. We Bayes-factora used 4 . ′ -UTR starts). Collapsed UTR regions with morewith regions UTR Collapsed starts). -UTR ′ UTRs. We generated one index on the basis of For HCMV mRNA abundance meas abundance mRNA HCMV For 6 1 with adaptations previously described ∆ γ Ψ treatment, 500 U or 25 ng/ml of ng/ml 25 or U 500 treatment, ) with an absolute value of at least 6 + Total RNA was isolated with 19 -containingRNA-seq reads ′ -UTR regions ( , 6 2 . Tandem. isoformUTR 2 8 . RT–PCR splicing Splicing-sensitive Supplementary h t t p Fig. 1 Fig. : / / w et al. w 2 w c 7 ------) , .

Lentiviral vector production and transduction. information is provided on the manufacturers’ websites. from Virusys, 1:1,000; and mouse UL83 (pp65) from Virusys, 1:5,000. Validationfrom Virusys, 1:5,000; mouse UL57 from Virusys, 1:1,000; mouse UL99 (pp28) β Laboratories, 1:2,000; CPEB1 (13583) from Cell Signaling Technologies, 1:1,000; CFI and (A301-096A)CSTF-77 (A301-092A), Antibodies to the following proteins were used at thethe first mock-infectedindicated sample (corresponding to dilutions:~100,000 cells total to per lane). normalized CSTF-64were amounts the type cell each within and Pierce), (Thermo assessment BCA by determined as content,protein total to according loaded 12,000 by clarified were lysates had been stored at −80 °C, with RIPA lysis buffer, on ice. After brief sonication, Westernanalysis. blot reactions are provided in Superscript III, Life Technologies). All primer sequences for RT–PCR and qPCR DNase treated, and only oligo(dT) was used for cDNA synthesis (generated with previouslyisoformsasperformed was described samples’ own poly(A) RNA-seq reads. qRT–PCR analysis of alternative the 3 of basis the on again condition,HSV-2 the for separately generated were to 2× in the NPC samples versus the HFF and EC libraries). Indexed annotations was sufficient for this analysis, and we had obtained a sequencing depth of 1.5× composite of infected HFF and EC poly(A) reads (neuronal versus non-neuronal poly(A) reads from the infected NPCs and generated another that was based on a P methods. Statistical was initiated 24 h after ASO transfection. 30 min at room temperature and added to the respective well. HCMV infection 100 well of a 12-well plate. For each ASO, 2 Antisense oligonucleotide (ASO) transfections. synthesized by IDT. consisting of known poly(A)-containing DNAs mixture spike-in 1% and (Illumina) library controlPhiX 10% with runs) end (Agilent) and sequenced on an Illumina MiSeq instrument (51 beads × AMPureXP 251-bpwith paired- purified was library The primer(s). reverse (barcode) IlluminaTruSeqand(Thermo) RNA ase smalluniversal forwardindexedand Superscript III (Invitrogen) and amplified by PCR with Phusion DNA polymer ligated to 5 (Thermo) and gel purified in the range of 250–750 nt. The purified RNAs were mide), run on a 6% polyacrylamide urea gel (NuPage), stained with SYBR gold T4-PNK reaction kit (NEB), eluted with 2× RNA Dynabeads loading dye (with 95% forma with down pulled M-280 streptavidin were (Invitrogen) RNAs magnetic fragmented beads, phosphorylated The on bead with (Ambion). a T1 forenrichto polyadenylated transcripts digestedandpartially RNasewaswith The RNA was ligated to 3 manufacturer’s the protocol. to Technologies)according (Life reagent with TRIzol cells from extracted was RNA total Briefly, modifications. minor with TAIL-seq. of drug selection. wasinitiated inthetransduced HFFs at 48h,and cells were harvested after d 3 MOI of 0.5 to 1, without polybrene or additional reagents. Puromycin selection 0.22- a through run and post-transfection h 60 at harvested wereSupernatants pMD2.G. psPAX2and format with 10 lentiviruses were generated in 293T cells with PEI transfection reagent, controlGFP and in UL99 CPEB1, sites.10-cmrestrictionNotI and SpeI the at inserted was CPEB1 cloned the and Technologies), (Life pCDNA3.1- from promoter CMV the CMV-turboGFPwithBiosystems)replaced was (Open pGIPZfrom PCR product was first cloned into the Topo-TAGCCCGCTGCAAAAATAGTG; vector pCR2.1 reverse,(Life Technologies).TCAGCAAGTGCAAAGGTGAC. The frame was PCR amplified from HFF cDNA with the following primers: forward, -actin (clone AC-15, A1978) from Sigma-Aldrich, 1:10,000; mouse IE (CH160) values were calculated with Student’swith calculated were values µ l of OptiMEM (Gibco) and 3 TAIL-seqpreviouslyChang byasperformed described was ′ adaptors (Illumina TruSeq small RNA kit), reverse transcribed with 7 cells per dish, with the second-generation packaging constructs The qPCRs were compared in a pairwise analysis, and analysis, pairwise a in compared were qPCRs The Whole cell lysates were prepared from cell pellets that pellets cell fromprepared werelysates cell Whole nature structural & molecular biology molecular & structural nature ′ µ ‘splint’ oligonucleotides (NNNGTCAGTTTTTTTTT) Supplementary Data Set 11 m filter. Freshly seeded HFFs were transduced at an attransduced were HFFs filter. Freshlym seeded g centrifugation for 10–15 min. Samples were Samples min. 10–15 for centrifugation µ l of RNAiMax (Life Sciences), incubated for µ l of 200- t test among technical replicates. The replicates. technical among test 2 The human CPEB1 open reading m 5 µ . All adapters and primers were -68 (A301-356A) from Bethyl Bethyl from (A301-356A) -68 1 5 × 104 HFFs were seeded per M stock ASO was mixed with 6 . 1 . µ . g of total RNAtotalfirstof was g et al.et ′ -UTR 2 5 - - ,

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. multiple hypothesis testing correction to estimate the FDR. Differential splicing Benjamini–Hochbergbyfollowed then was andexon-junction fragmentsand exon both changessplicingwith of significance statistical the evaluateto used or skipping isoform of each cassette exon was counted, and Fisher’snumber of exonexact or exon-junctiontest fragments wasuniquely supporting the inclusion andincreased the statistical power in detecting splicing changes. The weighted exons,cassettefor especially junctions,supportingexonfragments of number probability score. This junction inference step substantiallya assigned was fragmentinferred estimated. Each was readspaired-end the of increased the effective origin actual the being isoform each of probability the and found, were ends tice given the fragment size), all possible isoforms (paths) between the anchored of reads that spanned one or more exons (up to three, which is sufficient in prac analysis, we followed the procedure described in Charizanis identifysignificant differences in expression, as previously described sample,and each within calculated were to repetitive elements was also performed. Gene-expression values (in RPKM (GenBank mapped to both the human genome (hg19 build) and the HCMV Merlin sion,readsforwereadaptor trimmed genome sequencesorlow-quality andthen bases APAnot.Foranthanexpresgene more occur timeslikelyto 10,000 switch is that indicates 10,000 of factor Bayes A 0.03. least at of valueabsolute an with etal. Katz by described asexpression, differential offavor in not versus offavor in data the of evidence the of weight representsthe factor Bayes The calculated. algorithmformedMISO thewith v0.5.2,and Bayes factorand mentioned in the respective figure legends. Genome-wideas replicates, technical APA the of error standardanalysis or s.d. wasas reported per are bars error nature structural & molecular biology molecular & structural nature 2 7 . We. Bayes-factora used threshold of 10,000 and difference values ( A Y 4 4 6 8 9 4 . 2 ) with GSNAP. Additional filtering of reads that mapped Z -score analysis was implementedwasto analysis -score et al. ∆ 1 9 Ψ . For each pair valueswere 6 4 .For AS ∆ Ψ 6 3 - - - ) )

64. 63. 62. 61. 60. Mann–Whitney test. Individual tail-length distributions were compared among samples with the bal distributions of these lengths were compared with the Kolmogorov–Smirnov genes with at least 20 mapped reads, median lengths were measured, and thegenes glo were counted. Reads with tails with lengths measuring 0 wereuniquely removed.mapped protein-coding Forgenes. For analysis of virally mapped reads, all the only obtain to filtered and features,human_herpesvirus_5_strain_Merlin with andannotations v19 gencode with Subread with assigned wereFeatures parameters. default STARunder with (human_herpesvirus_5_strain_Merlin) read.Reads were aligned against the human genome (hg19) and viral genomes paired fastq files corresponding to the 5 (ref. tailseeker2 instrumenton analysis,wereMiSeq andrun fromdownloadedtheimage files events were identified by requiring FDR <0.05 and |

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