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Human Cytomegalovirus Induces and Exploits Roquin to Counteract the IRF1-Mediated Antiviral State

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Human Cytomegalovirus Induces and Exploits Roquin to Counteract the IRF1-Mediated Antiviral State Human cytomegalovirus induces and exploits Roquin to counteract the IRF1-mediated antiviral state Jaewon Songa,b,1, Sanghyun Leea,b,1, Dong-Yeon Choa,b, Sungwon Leea,b, Hyewon Kima,b, Namhee Yuc,d, Sanghyuk Leec,d, and Kwangseog Ahna,b,2 aSchool of Biological Sciences, Seoul National University, 08826 Seoul, Republic of Korea; bCenter for RNA Research, Institute for Basic Science, 08826 Seoul, Republic of Korea; cDepartment of Life Science, Ewha Womans University, 03760 Seoul, Republic of Korea; and dEwha Research Center for Systems Biology, Ewha Womans University, 03760 Seoul, Republic of Korea Edited by Peter A. Barry, University of California, Davis, CA, and accepted by Editorial Board Member Carl F. Nathan August 3, 2019 (received for review June 3, 2019) RNA represents a pivotal component of host–pathogen interac- mRNAs of cellular chemokines and cytokines and posttranscrip- tions. Human cytomegalovirus (HCMV) infection causes extensive tionally repress their expression. We previously reported that the alteration in host RNA metabolism, but the functional relationship HCMV-encoded miRNA miR-UL148D down-regulates the between the virus and cellular RNA processing remains largely cellular chemokine RANTES (20). Comprehensive studies of viral unknown. Through loss-of-function screening, we show that miRNAs revealed that they inhibit cytokine signaling, trafficking, HCMV requires multiple RNA-processing machineries for efficient and release, which strongly suppresses the host immune response viral lytic production. In particular, the cellular RNA-binding pro- (21, 22). tein Roquin, whose expression is actively stimulated by HCMV, During HCMV lytic infection, host cells are influenced by plays an essential role in inhibiting the innate immune response. certain unique features associated with RNA processing. In Transcriptome profiling revealed Roquin-dependent global down- contrast to other viruses that induce shutdown of host mRNA regulation of proinflammatory cytokines and antiviral genes in translation, HCMV stimulates this activity by blocking protein HCMV-infected cells. Furthermore, using cross-linking immunopre- kinase R activation (23) and modulates cellular RNA-processing cipitation (CLIP)-sequencing (seq), we identified IFN regulatory fac- machinery to accelerate viral gene translation (24–26). Addition- IRF1 tor 1 ( ), a master transcriptional activator of immune responses, ally, viral mRNAs undergo extensive posttranscriptional modifi- MICROBIOLOGY as a Roquin target gene. Roquin reduces IRF1 expression by directly cations, such as alternative splicing and polyadenylation (27, 28), binding to its mRNA, thereby enabling suppression of a variety of with elevated expressions of proteins regulating such modifications antiviral genes. This study demonstrates how HCMV exploits host during HCMV infection (29, 30). Although these reports reveal RNA-binding protein to prevent a cellular antiviral response and that HCMV might require cellular RNA-processing machinery for offers mechanistic insight into the potential development of CMV efficient replication at multiple steps, how these RNA-binding pro- therapeutics. teins regulate host mRNAs and cellular functions remains largely unexplored. human cytomegalovirus | RNA-binding protein | immune evasion | In this study, we examined the role of cellular RNA-processing proinflammatory cytokine proteins in immune regulation during HCMV lytic production by employing loss-of-function screening. We found that 19 genes uman cytomegalovirus (HCMV) is a member of the Hβ-herpesvirus family and a ubiquitous pathogen that estab- Significance lishes lifelong latency in large populations worldwide (1). During primary infection of HCMV, which rarely exhibits pathogenic Human cytomegalovirus (HCMV) is a major cause of birth de- symptoms in healthy humans, the virus overcomes innate and fects and diseases in immune-compromised patients. HCMV is adaptive immune responses in the host and establishes permanent able to infect and establish latency in large populations by latency. Reactivation from latency can be dangerous in immune- employing multiple strategies to evading the host immune compromised hosts, with HCMV congenital infection being a response. Here, we report that HCMV suppresses cytokine- leading cause of neurological defects in infants (2–4). Currently, mediated antiviral responses by increasing the expression of there is no therapeutic intervention capable of curing chronic cellular RNA-binding protein, Roquin. We show that Roquin HCMV infection, and no vaccines are available to prevent HCMV inhibits cytokine production by directly binding to cytokine infection. This is largely because of the multiple strategies employed mRNAs and by repressing the expression of their transcription by HCMV to evade innate and adaptive immunity. Thus, having a activator. This study highlights that cellular RNA metabolism, thorough understanding of how the virus evades the host immune which is controlled by HCMV for immune evasion, can be the system is necessary for the development of anti-HCMV vaccines target for developing anti-HCMV therapeutics. and therapeutics. Primary HCMV infection provokes a robust innate immune Author contributions: J.S., Sanghyun Lee, and K.A. designed research; J.S., Sanghyun Lee, Sungwon Lee, and H.K. performed research; J.S., Sanghyun Lee, D.-Y.C., N.Y., and response in terms of rapid induction of nuclear factor kappaB Sanghyuk Lee analyzed data; and J.S., Sanghyun Lee, and K.A. wrote the paper. κ (NF- B)- and IFN regulatory factor (IRF)3-signaling pathways The authors declare no conflict of interest. – in cells (5 7). Proinflammatory cytokines, type I interferons, and This article is a PNAS Direct Submission. P.A.B. is a guest editor invited by the IFN-stimulated genes (ISGs) are produced and confer antiviral Editorial Board. effects by targeting various steps of HCMV replication (8, 9). Published under the PNAS license. However, HCMV has evolved strategies of regulating cellular Data deposition: The data reported in this paper have been deposited in the Gene Ex- cytokines in order to counteract the host immune system. First, pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE132081). HCMV encodes cellular cytokine mimics and decoy receptors in 1J.S. and Sanghyun Lee contributed equally to this work. order to modulate cytokine activity and restrict cell migration 2To whom correspondence may be addressed. Email: [email protected]. – and differentiation (10 15). Second, HCMV inhibits production This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of cytokines by blocking the activation of transcription factors 1073/pnas.1909314116/-/DCSupplemental. (16–19). Third, viral microRNAs (miRNAs) directly target the Published online August 26, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1909314116 PNAS | September 10, 2019 | vol. 116 | no. 37 | 18619–18628 Downloaded by guest on September 27, 2021 are required for HCMV lytic production, among which Roquin Results was critical for viral gene expression. HCMV-induced Roquin HCMV Requires Multiple Cellular RNA-Processing Activities for Its played a dominant role in the suppression of cytokine production. Efficient Lytic Production. HCMV harbors multiple viral genes During viral infection, Roquin directly bound to and regulated that modulate cellular mRNA processing, with some host cell expression of cellular RNA transcripts including those of cytokine genes capable of positively or negatively regulating viral infec- genes and IRF1, which was crucial for increased cytokine sup- tion. To identify genes essential for HCMV lytic production, an pression and enhanced viral replication. These results demon- RNA interference (RNAi)-screening experiment was performed strated areas of HCMV evolution to exploit host RNA-binding using 687 genes involved in cellular RNA processing. The small- proteins in order to modulate the cellular environment in its favor. interfering RNA (siRNA) library contained three gene categories: RNA-Binding protein A 409 genes Total 687 genes 366 siRNA HCMV infection 41 1 1 C. elegans 213 6 59 48 h 48 h Measure RNA level ortholog RNase of viral genes 67 genes 245 genes Primary HFFs B IE1 (Immediate early) C 4 IE1 UL44UL99 2 CCT8 RC3H1 1 SNRPF 0 CSDC2 ZC3H4 -1 YTHDC1 -2 RBM23 HCMV mRNA 2 -3 RBM4 Log (siTarget/siControl) KIAA0391 -4 ZCCHC16 -5 RNASE13 GPKOW UL44 (Early) EIF3B 2 HNRNPC TRA2A 1 HNRNPM 0 PSMA3 SUPT6H -1 URI1 RBM43 -2 YBEY HCMV mRNA 2 -3 PABPC3 RBM46 Log (siTarget/siControl) -4 MBNL3 RPL21 -5 YTHDF1 UL99 (Late) ASMTL 2 EIF3G SNRPC 1 CSE1L RBMX 0 PSMB6 -1 SPINT2 PSMB3 -2 PSMC2 HCMV mRNA 2 -3 POLR2G log2(Fold Change) Log (siTarget/siControl) -4 -5 −440 D HCMV HSV-1 108 107 106 105 104 103 Titer (PFU/mL) 102 101 100 siRNA URI1 CCT8 YBEY RBM4 EIF3B EIF3G ZC3H4 CSE1L Control TRA2A RC3H1 ASMTL RBM46 RBM23 MBNL3 RBM43 SNRPF CSDC2 PSMB3 PSMA3 PSMB6 PSMC2 SNRPC GPKOW PABPC3 POLR2G HNRNPC HNRNPM KIAA0391 RNASE13 ZCCHC16 Fig. 1. Identification of RNA-binding proteins that regulate HCMV lytic infection through siRNA screening. (A) Experimental scheme of siRNA screening. RNases, RNA-binding proteins, and orthologous genes in the small-RNA pathway from C. elegans were collected for screening. HFF cells underwent two rounds of siRNA transfection, with infection with HCMV (MOI = 2). Viral gene expression was measured by qRT-PCR at 48 hpi and normalized against GAPDH mRNA levels. (B) Summarized results of RNAi screening. IE1, UL44, and UL99 mRNA levels were quantified.
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