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• W2913600982 abstract "Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA–protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3′ terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3′UTR consensus sequences. During RV infection, ATP5B bound to the RV 3′UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis. Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA–protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3′ terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3′UTR consensus sequences. During RV infection, ATP5B bound to the RV 3′UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis. Viruses are obligate intracellular pathogens that usurp host cellular machinery for efficient replication and production of progeny infectious particles. Despite the availability of multiple genetic and biochemical tools that enable the examination of rotavirus–host interactions in a unbiased manner, including genome-wide siRNA screens (1Green V.A. Pelkmans L. A systems survey of progressive host-cell reorganization during rotavirus infection.Cell Host Microbe. 2016; 20 (27414499): 107-12010.1016/j.chom.2016.06.005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 2Silva-Ayala D. López T. Gutiérrez M. Perrimon N. López S. Arias C.F. Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23733942): 10270-1027510.1073/pnas.1304932110Crossref PubMed Scopus (60) Google Scholar), interactome analysis of protein–protein interactions (3Ding S. Mooney N. Li B. Kelly M.R. Feng N. Loktev A.V. Sen A. Patton J.T. Jackson P.K. Greenberg H.B. Comparative proteomics reveals strain-specific β-TrCP degradation via rotavirus NSP1 hijacking a host cullin–3-Rbx1 complex.PLoS Pathog. 2016; 12 (27706223): e100592910.1371/journal.ppat.1005929Crossref PubMed Scopus (42) Google Scholar), and the recently developed CRISPR-Cas9 screens (4Ding S. Diep J. Feng N. Ren L. Li B. Ooi Y.S. Wang X. Brulois K.F. Yasukawa L.L. Li X. Kuo C.J. Solomon D.A. Carette J.E. Greenberg H.B. STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection.Nat. Commun. 2018; 9 (29662124): 148510.1038/s41467-018-03782-zCrossref PubMed Scopus (48) Google Scholar), the molecular interactions between rotaviral RNA and host proteins remain relatively poorly understood. RNA-binding proteins play many important roles in cellular functions (5Hentze M.W. Castello A. Schwarzl T. Preiss T. A brave new world of RNA-binding proteins.Nat. Rev. Mol. Cell Biol. 2018; 19 (29339797): 327-34110.1038/nrm.2017.130Crossref PubMed Scopus (703) Google Scholar). With the exception of cytoplasmic RNA sensors, RIG-I–like receptors, including RIG-I, MDA5, and LGP2, and effector molecules such as RNase L and protein kinase R (6Loo Y.M. Gale Jr., M. Immune signaling by RIG-I–like receptors.Immunity. 2011; 34 (21616437): 680-69210.1016/j.immuni.2011.05.003Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar7Sánchez-Tacuba L. Rojas M. Arias C.F. López S. Rotavirus controls activation of the 2′–5′-oligoadenylate synthetase/RNase L pathway using at least two distinct mechanisms.J. Virol. 2015; 89 (26401041): 12145-1215310.1128/JVI.01874-15Crossref PubMed Scopus (28) Google Scholar, 8Silverman R.H. Viral encounters with 2′,5′-oligoadenylate synthetase and RNase L during the interferon antiviral response.J. Virol. 2007; 81 (17804500): 12720-1272910.1128/JVI.01471-07Crossref PubMed Scopus (436) Google Scholar9Uzri D. Greenberg H.B. Characterization of rotavirus RNAs that activate innate immune signaling through the RIG-I–like receptors.PLoS One. 2013; 8 (23894547): e6982510.1371/journal.pone.0069825Crossref PubMed Scopus (32) Google Scholar), the identities of host proteins that specifically bind to virus RNA genome or other replication intermediates and by-products, have rarely been characterized. Lately, interrogation of stem-loop structures within the untranslated regions (UTRs) of Zika virus and dengue virus has led to the discovery of host proteins Musashi-1 and TRIM25 that play critical roles in the replication cycle of these flaviviruses (10Chavali P.L. Stojic L. Meredith L.W. Joseph N. Nahorski M.S. Sanford T.J. Sweeney T.R. Krishna B.A. Hosmillo M. Firth A.E. Bayliss R. Marcelis C.L. Lindsay S. Goodfellow I. Woods C.G. Gergely F. Neurodevelopmental protein Musashi-1 interacts with the Zika genome and promotes viral replication.Science. 2017; 357 (28572454): 83-8810.1126/science.aam9243Crossref PubMed Scopus (106) Google Scholar, 11Manokaran G. Finol E. Wang C. Gunaratne J. Bahl J. Ong E.Z. Tan H.C. Sessions O.M. Ward A.M. Gubler D.J. Harris E. Garcia-Blanco M.A. Ooi E.E. Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness.Science. 2015; 350 (26138103): 217-22110.1126/science.aab3369Crossref PubMed Scopus (263) Google Scholar). It was also found that, for influenza virus and coxsackievirus, defective RNA secondary structures markedly diminish binding of host exosome complex and PCBP2, respectively, and result in reduced replication (12Lévêque N. Garcia M. Bouin A. Nguyen J.H.C. Tran G.P. Andreoletti L. Semler B.L. Functional consequences of RNA 5′-terminal deletions on coxsackievirus B3 RNA replication and ribonucleoprotein complex formation.J. Virol. 2017; 91 (28539455): e00423-e00517Crossref PubMed Scopus (23) Google Scholar, 13Rialdi A. Hultquist J. Jimenez-Morales D. Peralta Z. Campisi L. Fenouil R. Moshkina N. Wang Z.Z. Laffleur B. Kaake R.M. McGregor M.J. Haas K. Pefanis E. Albrecht R.A. Pache L. Chanda S. Jen J. Ochando J. Byun M. Basu U. Garcia-Sastre A. Krogan N. van Bakel H. Marazzi I. The RNA exosome syncs IAV-RNAPII transcription to promote viral ribogenesis and infectivity.Cell. 2017; 169 (28475896): 679-692.e1410.1016/j.cell.2017.04.021Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Rotaviruses (RVs) 4The abbreviations used are: RVrotavirusRaPIDRNA–protein interaction detectionm.o.i.multiplicity of infectionqPCRquantitative PCRGAPDHglyceraldehyde-3-phosphate dehydrogenaseDAPI4′,6-diamidino-2-phenylindoleIPimmunoprecipitationDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumFISHfluorescent in situ hybridizationRMSDroot-mean-square deviationIECintestinal epithelial cellSAINTSignificance Analysis of INTeractomehpihours post-infectionMAVSmitochondrial antiviral-signaling proteinVSVvesicular stomatitis virus. are nonenveloped, segmented dsRNA viruses in the Reoviridae family (14Estes M.K.G. HB et al.Knipe D.M. Howley P.M. Rotaviruses. 6th Ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, PA2013: 1347-1401Google Scholar). RV infections are the leading cause of infantile diarrhea and severe gastroenteritis, resulting in around 215,000 deaths annually worldwide (15Tate J.E. Burton A.H. Boschi-Pinto C. Parashar U.D. World Health Organization–Coordinated Global Rotavirus Surveillance NetworkGlobal, regional, and national estimates of rotavirus mortality in children <5 years of age, 2000–2013.Clin. Infect. Dis. 2016; 62 (27059362): S96-S10510.1093/cid/civ1013Crossref PubMed Scopus (722) Google Scholar). Similar to those of influenza virus and orthoreovirus, RV gene segments have to be precisely organized and packaged into newly synthesized icosahedral virus particles during genome assembly (16Long C.P. McDonald S.M. Rotavirus genome replication: some assembly required.PLoS Pathog. 2017; 13 (28426777): e100624210.1371/journal.ppat.1006242Crossref PubMed Scopus (18) Google Scholar). A previous study of a highly-related bluetongue virus and recent studies of RV suggest that the smaller RV RNA segments may be sorted into the newly forming virion particles first, and then the larger RNA segments enter the particle via RNA–RNA interactions in a sequential and NSP2-dependent manner (17Fajardo Jr, T. Sung P.Y. Roy P. Disruption of specific RNA–RNA interactions in a double-stranded RNA virus inhibits genome packaging and virus infectivity.PLoS Pathog. 2015; 11 (26646790): e100532110.1371/journal.ppat.1005321Crossref PubMed Scopus (28) Google Scholar, 18Fajardo T. Sung P.Y. Celma C.C. Roy P. Rotavirus genomic RNA complex forms via specific RNA–RNA interactions: disruption of RNA complex inhibits virus infectivity.Viruses. 2017; 9 (28661470): 16710.3390/v9070167Crossref Scopus (16) Google Scholar19Borodavka A. Dykeman E.C. Schrimpf W. Lamb D.C. Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments.eLife. 2017; 6 (28922109): e2745310.7554/eLife.27453Crossref PubMed Scopus (42) Google Scholar). Hydrodynamic studies that examined the “stiffness” of RV RNA segments in vitro also suggested that packaging of the viral RNA segments into the capsid likely necessitates intimate RNA–protein interactions (20Kapahnke R. Rappold W. Desselberger U. Riesner D. The stiffness of dsRNA: hydrodynamic studies on fluorescence-labeled RNA segments of bovine rotavirus.Nucleic Acids Res. 1986; 14 (3010231): 3215-322810.1093/nar/14.8.3215Crossref PubMed Scopus (24) Google Scholar). Given the precision required and the likely high-energy consumption needed for RNA packaging, we hypothesize that host RNA-binding proteins contribute to the correct assembly of RV gene segments. rotavirus RNA–protein interaction detection multiplicity of infection quantitative PCR glyceraldehyde-3-phosphate dehydrogenase 4′,6-diamidino-2-phenylindole immunoprecipitation Dulbecco's modified Eagle's medium fetal bovine serum fluorescent in situ hybridization root-mean-square deviation intestinal epithelial cell Significance Analysis of INTeractome hours post-infection mitochondrial antiviral-signaling protein vesicular stomatitis virus. For most organisms, ATP hydrolysis powered by the ATPase machinery is the single most important “energy currency” (21Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.Nature. 1994; 370 (8065448): 621-62810.1038/370621a0Crossref PubMed Scopus (2749) Google Scholar, 22Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Direct observation of the rotation of F1-ATPase.Nature. 1997; 386 (9069291): 299-30210.1038/386299a0Crossref PubMed Scopus (1962) Google Scholar). For bacteriophages and large DNA viruses like herpesvirus and poxviruses, a central component of the packaging motor that drives viral genome assembly is the ATPase subunit, provided by the viruses themselves (23Hilbert B.J. Hayes J.A. Stone N.P. Duffy C.M. Sankaran B. Kelch B.A. Structure and mechanism of the ATPase that powers viral genome packaging.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (26150523): E3792-E379910.1073/pnas.1506951112Crossref PubMed Scopus (50) Google Scholar, 24Nadal M. Mas P.J. Blanco A.G. Arnan C. Solà M. Hart D.J. Coll M. Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20805464): 16078-1608310.1073/pnas.1007144107Crossref PubMed Scopus (89) Google Scholar25Rao V.B. Feiss M. The bacteriophage DNA packaging motor.Annu. Rev. Genet. 2008; 42 (18687036): 647-68110.1146/annurev.genet.42.110807.091545Crossref PubMed Scopus (290) Google Scholar). In contrast, RNA viruses, except bluetongue virus (26Stäuber N. Martinez-Costas J. Sutton G. Monastyrskaya K. Roy P. Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates.J. Virol. 1997; 71 (9311795): 7220-7226Crossref PubMed Google Scholar), by and large harbor a relative small genome and rarely encode viral ATPases (27Rodríguez P.L. Carrasco L. Poliovirus protein 2C has ATPase and GTPase activities.J. Biol. Chem. 1993; 268 (8385138): 8105-8110Abstract Full Text PDF PubMed Google Scholar), inviting the question whether or not these viruses hijack the host ATPase complex as an alternative strategy to obtain energy for genome packaging. Here, we employed a novel and powerful technique named RNA-Protein Interaction Detection (RaPID), recently developed to study RNA–protein interactions (28Ramanathan M. Majzoub K. Rao D.S. Neela P.H. Zarnegar B.J. Mondal S. Roth J.G. Gai H. Kovalski J.R. Siprashvili Z. Palmer T.D. Carette J.E. Khavari P.A. RNA–protein interaction detection in living cells.Nat. Methods. 2018; 15 (29400715): 207-21210.1038/nmeth.4601Crossref PubMed Scopus (154) Google Scholar), to comprehensively profile the host factors that bind to a stretch of conserved sequences within 3′UTR of group A RV genomes. Surprisingly, we identified ATP5B, an integral part of the mitochondrial F1–F0-ATPase complex (29von Ballmoos C. Cook G.M. Dimroth P. Unique rotary ATP synthase and its biological diversity.Annu. Rev. Biophys. 2008; 37 (18573072): 43-6410.1146/annurev.biophys.37.032807.130018Crossref PubMed Scopus (145) Google Scholar), as a cellular component that co-precipitated and co-localized with RV dsRNA during infection. Functional dissection using small interfering RNA (siRNA) knockdown and a panel of small-molecule pharmacological inhibitors suggested that ATP5B assists RV genome replication and virion assembly. Thus, our study systematically interrogated the host proteins that interact with the RV 3′ terminus and revealed a tractable method to rapidly identify host proteins that bind to viral RNA sequences of interest in living cells. For group A human and animal RVs, the last seven nucleotides within the mRNA 3′UTR, 5′-UGUGACC-3′, are highly conserved in all 11 gene segments (30Chen D. Patton J.T. De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation.RNA. 2000; 6 (11073221): 1455-146710.1017/S1355838200001187Crossref PubMed Scopus (53) Google Scholar, 31Patton J.T. Rotavirus VP1 alone specifically binds to the 3′ end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis.J. Virol. 1996; 70 (8892917): 7940-7947Crossref PubMed Google Scholar32Wentz M.J. Patton J.T. Ramig R.F. The 3′-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis.J. Virol. 1996; 70 (8892905): 7833-7841Crossref PubMed Google Scholar) and distinct from those in group B (5′-UAUACCC-3′) and group C (5′-UGUGGCU-3′) RVs (Fig. 1). This short sequence forms a cis-acting signal that contributes to the efficient synthesis of minus strand RNA and is also conducive to viral gene expression in the host cells through interaction with RV protein NSP3 (33Patton J.T. Wentz M. Xiaobo J. Ramig R.F. cis-Acting signals that promote genome replication in rotavirus mRNA.J. Virol. 1996; 70 (8648733): 3961-3971Crossref PubMed Google Scholar, 34Poncet D. Laurent S. Cohen J. Four nucleotides are the minimal requirement for RNA recognition by rotavirus nonstructural protein NSP3.EMBO J. 1994; 13 (8076612): 4165-417310.1002/j.1460-2075.1994.tb06734.xCrossref PubMed Scopus (68) Google Scholar). However, the nature and identity of host factors that bind to this important RNA region remain unknown. To comprehensively identify host proteins that interact with RV 3′UTR consensus sequence, we took advantage of a new screening approach, named RaPID, that detects, with high sensitivity, any protein in the vicinity of the target RNA molecule (28Ramanathan M. Majzoub K. Rao D.S. Neela P.H. Zarnegar B.J. Mondal S. Roth J.G. Gai H. Kovalski J.R. Siprashvili Z. Palmer T.D. Carette J.E. Khavari P.A. RNA–protein interaction detection in living cells.Nat. Methods. 2018; 15 (29400715): 207-21210.1038/nmeth.4601Crossref PubMed Scopus (154) Google Scholar). In brief, biotin–protein ligase BirA (36Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.J. Cell Biol. 2012; 196 (22412018): 801-81010.1083/jcb.201112098Crossref PubMed Scopus (1234) Google Scholar) is N-terminally fused to a 22-amino acid λN peptide that recognizes bacteriophage λ BoxB stem-loops with high affinity (37Austin R.J. Xia T. Ren J. Takahashi T.T. Roberts R.W. Designed arginine-rich RNA-binding peptides with picomolar affinity.J. Am. Chem. Soc. 2002; 124 (12224929): 10966-1096710.1021/ja026610bCrossref PubMed Scopus (72) Google Scholar), flanking both 5′ and 3′ ends of an RNA of interest (Fig. 2A). Thus, BirA ligase is brought in close proximity to and biotinylates all proteins that directly or indirectly associate with target RNA. We then perform immunoprecipitation with streptavidin beads and used MS and bioinformatics analysis to identify interacting protein partners. As a proof of principle, we first tested the interaction between a pair of positive controls: a 15-nucleotide UG–rich EDEN15 motif (UGUUUGUUUGUUUGU) that is reported to bind to the CUG triplet repeat RNA-binding protein 1 (CUG-BP1) (38Graindorge A. Le Tonquèze O. Thuret R. Pollet N. Osborne H.B. Audic Y. Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis.Nucleic Acids Res. 2008; 36 (18267972): 1861-187010.1093/nar/gkn031Crossref PubMed Scopus (42) Google Scholar). As expected, specific immunoprecipitation of endogenous CUG-BP1 was only observed with EDEN15 and not with the negative control scrambled sequences (Fig. 2B). Next, we examined the well-documented interaction between the conserved RV 3′UTR and an RV nonstructural protein 3 (NSP3). Consistent with previous reports (34Poncet D. Laurent S. Cohen J. Four nucleotides are the minimal requirement for RNA recognition by rotavirus nonstructural protein NSP3.EMBO J. 1994; 13 (8076612): 4165-417310.1002/j.1460-2075.1994.tb06734.xCrossref PubMed Scopus (68) Google Scholar), we detected strong binding of the UGUGACC sequence with either ectopically expressed GFP-tagged NSP3 or endogenous NSP3 expressed during RV infection (Fig. 2, C and D), suggesting that this technique is well-suited to interrogate the host protein–binding partners of the RV 3′UTR sequences. In addition to examining binding proteins of 3′UTR RV monomers, we further tested whether a pentamer of RV 3′UTRs with two adenosine spacers between monomers would increase the sensitivity for detecting host protein binding to NSP3. However, our results indicated that a monomer was equally effective for pulling down NSP3 (data not shown). Therefore, for the rest of the study, we used the monomeric probes to reduce the possibility of creating artificial secondary structures. By comparing a scrambled sequence (AUAGGCGUC) to an authentic conserved RV 3′UTR probe (GAUGUGACC) in the presence or absence of RV infection, we were able to identify host proteins that specifically immunoprecipitated with the 3′UTR consensus sequences (Table S1). We then used CRAPome filtering analysis (39Mellacheruvu D. Wright Z. Couzens A.L. Lambert J.P. St-Denis N.A. Li T. Miteva Y.V. Hauri S. Sardiu M.E. Low T.Y. Halim V.A. Bagshaw R.D. Hubner N.C. Al-Hakim A. Bouchard A. Faubert D. Fermin D. Dunham W.H. Goudreault M. Lin Z.Y. Badillo B.G. Pawson T. Durocher D. Coulombe B. Aebersold R. Superti-Furga G. Colinge J. Heck A.J. Choi H. Gstaiger M. Mohammed S. Cristea I.M. Bennett K.L. Washburn M.P. Raught B. Ewing R.M. Gingras A.C. Nesvizhskii A.I. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data.Nat. Methods. 2013; 10 (23921808): 730-73610.1038/nmeth.2557Crossref PubMed Scopus (912) Google Scholar) and a Significance Analysis of INTeractome (SAINT) threshold score of 0.9 to identify specific host proteins. Our analysis identified three high-confidence “hits”: the mitochondrial F1 complex β-polypeptide ATP5B; the RAS oncogene family member RAB1A; and the isoleucyl-tRNA synthetase IARS as interacting partners to the RV 3′UTR consensus sequences (Fig. 2E and Table S2). Of note, ATP5B, an ATP synthase β subunit (29von Ballmoos C. Cook G.M. Dimroth P. Unique rotary ATP synthase and its biological diversity.Annu. Rev. Biophys. 2008; 37 (18573072): 43-6410.1146/annurev.biophys.37.032807.130018Crossref PubMed Scopus (145) Google Scholar), was found to be enriched at 100-fold in its interaction with 3′UTR probe over control (Fig. 2E). ATP5B was previously reported to promote human immunodeficiency virus (HIV)-1 replication and chikungunya virus infection (40Zhou H. Xu M. Huang Q. Gates A.T. Zhang X.D. Castle J.C. Stec E. Ferrer M. Strulovici B. Hazuda D.J. Espeseth A.S. Genome-scale RNAi screen for host factors required for HIV replication.Cell Host Microbe. 2008; 4 (18976975): 495-50410.1016/j.chom.2008.10.004Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 41Fongsaran C. Jirakanwisal K. Kuadkitkan A. Wikan N. Wintachai P. Thepparit C. Ubol S. Phaonakrop N. Roytrakul S. Smith D.R. Involvement of ATP synthase β subunit in chikungunya virus entry into insect cells.Arch. Virol. 2014; 159 (25168043): 3353-336410.1007/s00705-014-2210-4Crossref PubMed Scopus (41) Google Scholar), prompting us to hypothesize that it may also act as a pro-RV host factor. Importantly, we also detected two other pivotal components of the ATP synthase complex, ATP5A1 and ATP5O, in the pulldown of 3′UTR sequence during RV infection (Fig. 2F). It is worth noting that all three ATP synthase subunits were found to interact with the RV 3′UTR probe only in the context of RV infection (Fig. 2F). Taken together, this new assay technique to detect host RNA-binding proteins allowed us to identify several novel proteins, including several ATP synthase subunits that had previously not been known to interact with viral RNA. We next carried out a set of experiments to validate the association between ATP5B and the conserved RV 3′UTR sequences. Using streptavidin beads to directly pull down biotinylated host proteins in mock- or RV-infected cells, we observed strong binding of endogenous ATP5B with an RV 3′UTR probe, but only very weak interaction was detected with the scrambled probe (Fig. 3A). Importantly, the ATP5B–3′UTR interaction was observed exclusively during active RV infection (Fig. 3A), suggesting that such interaction might be regulated by other host factors, viral factors, or virus-induced cellular changes. We hypothesize that to physically interact with RV RNA, ATP5B is likely to co-localize with viral RNA in infected cells. We first determined ATP5B localization in the presence or absence of RV infection. In mock-infected cells, almost all ATP5B was found at the mitochondria (Fig. 3B, upper panel), consistent with previous reports (42Yang W. Nagasawa K. Münch C. Xu Y. Satterstrom K. Jeong S. Hayes S.D. Jedrychowski M.P. Vyas F.S. Zaganjor E. Guarani V. Ringel A.E. Gygi S.P. Harper J.W. Haigis M.C. Mitochondrial sirtuin network reveals dynamic SIRT3-dependent deacetylation in response to membrane depolarization.Cell. 2016; 167 (27881304): 985-1000.e2110.1016/j.cell.2016.10.016Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and its role as part of the ATP synthase complex. However, in RV-infected cells, we observed a significant condensation of mitochondrial organization in viral antigen VP6–positive cells (Fig. 3B, lower panel), recently noted by Green and Pelkmans (1Green V.A. Pelkmans L. A systems survey of progressive host-cell reorganization during rotavirus infection.Cell Host Microbe. 2016; 20 (27414499): 107-12010.1016/j.chom.2016.06.005Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) as well. To examine the subcellular localization of ATP5B relative to viral RNA, we designed specific fluorescent in situ hybridization (FISH) probes that detect RV RNA with high resolution. Our RV FISH probes were highly specific for viral RNA and only observed within the viroplasm, the distinct punctate viral factories that are positive for the nonstructural protein NSP2 (Fig. 3C). Importantly, viral RNA signal was also seen, at least partially, to overlap with ATP5B localization during infection (Fig. 3D and inset). In contrast, minimal co-localization was observed between RV RNA with mitochondrial antiviral-signaling protein (MAVS) (Fig. 3D). Therefore, data from both immunoprecipitation and immunofluorescence experiments are consistent with our screening results indicating that ATP5B is a bona fide RV 3′UTR–interacting protein. Having established ATP5B–rotaviral RNA interaction, we next sought to examine the functional role of ATP5B during RV infection. Because genetic depletion of ATP5B induces lethality (43Blomen V.A. Majek P. Jae L.T. Bigenzahn J.W. Nieuwenhuis J. Staring J. Sacco R. van Diemen F.R. Olk N. Stukalov A. Marceau C. Janssen H. Carette J.E. Bennett K.L. Colinge J. Superti-Furga G. Brummelkamp T.R. Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350 (26472760): 1092-109610.1126/science.aac7557Crossref PubMed Scopus (503) Google Scholar), we utilized siRNA to knock down ATP5B expression levels. All three top candidates identified in our proteomics screen, ATP5B, RAB1A, and IARS, were effectively silenced with their specific siRNAs and examined by their mRNA levels by quantitative PCR (Fig. 4A). Both ATP5B and IARS siRNA also led to a marked reduction of respective protein levels, although the decrease was more significant with IARS than ATP5B (Fig. 4B). Importantly, in a multiple-round infection assay, at 24 h post-infection, knockdown of ATP5B led to 40% reduction in the mRNA levels of NSP5, an RV gene transcript representative of intracellular RV RNA synthesis (Fig. 4C), and we also observed an ∼40% decrease in the amount of infectious RVs in the supernatant of the ATP5B knockdown cells (Fig. 4D), suggesting that ATP5B facilitates RV replication in host cells. Given that all group A RVs share consensus sequences of the ATP5B-binding site at their 3′UTR ends (Fig. 1), we next examined whether the pro-RV role of ATP5B is conserved across different RV strains. Consistent with our hypothesis, the infectivity of a panel of cell culture adapted group A human and animal RVs was inhibited by siRNA depletion of ATP5B (Fig. 4E). In contrast, the replication of several other RNA viruses, including vesicular stomatitis virus and two strains of influenza A viruses, which do not possess 3′-UGUGACC sequences, was not affected by ATP5B knockdown (Fig. 4, F and G). ATP5B knockdown also did not influence the replication of Salmonella typhimurium, an intracellular bacterial pathogen or even reovirus, another Reoviridae family member with many properties similar to RVs (Fig. 4, H and I), thereby in support of a highly-specific pro-RV role of ATP5B. To mechanistically determine at which step in the RV replication cycle in which ATP5B is involved, we undertook a series of experiments to target various molecular processes within a single RV replication cycle. First, initial viral adsorption and subsequent endocytosis, assayed by input viral RNA levels 4 and 37 °C post-incubation, respectively, and using EDTA wash as a negative control, were not affected by ATP5B siRNA, when compared with control siRNA (Fig. 5, A and B). These two pieces of experimental evidence strongly suggest that early events in RV infection are not perturbed by the loss of ATP5B. Next, we measured viral RNA by quantitative PCR and protein levels using polyclonal antibody that recognizes RV double-layered particles (44Feng N. Vo P.T. Chung D. Vo T.V. Hoshino Y. Greenberg H.B. Heterotypic protection following oral immunization with live heterologous rotaviruses in a mouse model.J. Infect. Dis. 1997; 175 (9203654): 330-34110.1093/infdis/175.2.330Cros" @default.
• W2913600982 created "2019-02-21" @default.
• W2913600982 creator A5010090782 @default.
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• W2913600982 date "2019-04-01" @default.
• W2913600982 modified "2023-10-18" @default.
• W2913600982 title "Profiling of rotavirus 3′UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication" @default.
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• W2913600982 cites W1556270435 @default.
• W2913600982 cites W1634155365 @default.
• W2913600982 cites W1827648715 @default.
• W2913600982 cites W1898313134 @default.
• W2913600982 cites W1910711340 @default.
• W2913600982 cites W1972964695 @default.
• W2913600982 cites W1973646620 @default.
• W2913600982 cites W1982514497 @default.
• W2913600982 cites W1987224659 @default.
• W2913600982 cites W1995623317 @default.
• W2913600982 cites W1995716654 @default.
• W2913600982 cites W2003907443 @default.
• W2913600982 cites W2004421522 @default.
• W2913600982 cites W2017579029 @default.
• W2913600982 cites W2027010869 @default.
• W2913600982 cites W2043305798 @default.
• W2913600982 cites W2047687916 @default.
• W2913600982 cites W2050832011 @default.
• W2913600982 cites W2052618584 @default.
• W2913600982 cites W2055066933 @default.
• W2913600982 cites W2058498150 @default.
• W2913600982 cites W2064917554 @default.
• W2913600982 cites W2068880841 @default.
• W2913600982 cites W2070096236 @default.
• W2913600982 cites W2071950147 @default.
• W2913600982 cites W2074033410 @default.
• W2913600982 cites W2077602890 @default.
• W2913600982 cites W2077614707 @default.
• W2913600982 cites W2094578393 @default.
• W2913600982 cites W2099860179 @default.
• W2913600982 cites W2100385339 @default.
• W2913600982 cites W2105707485 @default.
• W2913600982 cites W2106615577 @default.
• W2913600982 cites W2120350522 @default.
• W2913600982 cites W2120382578 @default.
• W2913600982 cites W2120975539 @default.
• W2913600982 cites W2123045377 @default.
• W2913600982 cites W2130963764 @default.
• W2913600982 cites W2131701481 @default.
• W2913600982 cites W2132771789 @default.
• W2913600982 cites W2132813363 @default.
• W2913600982 cites W2134144300 @default.
• W2913600982 cites W2147021126 @default.
• W2913600982 cites W2155831456 @default.
• W2913600982 cites W2157729395 @default.
• W2913600982 cites W2164709597 @default.
• W2913600982 cites W2166343003 @default.
• W2913600982 cites W2167844024 @default.
• W2913600982 cites W2169659701 @default.
• W2913600982 cites W2174686320 @default.
• W2913600982 cites W2195485567 @default.
• W2913600982 cites W2204067734 @default.
• W2913600982 cites W2228688349 @default.
• W2913600982 cites W2300378039 @default.
• W2913600982 cites W2328134362 @default.
• W2913600982 cites W2501188336 @default.
• W2913600982 cites W2528348911 @default.
• W2913600982 cites W2544727711 @default.
• W2913600982 cites W2556949830 @default.
• W2913600982 cites W2606200245 @default.
• W2913600982 cites W2607328645 @default.
• W2913600982 cites W2611318506 @default.
• W2913600982 cites W2615136515 @default.
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• W2913600982 cites W2724095907 @default.
• W2913600982 cites W2748927955 @default.
• W2913600982 cites W2785329615 @default.
• W2913600982 cites W2786230719 @default.
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• W2913600982 doi "https://doi.org/10.1074/jbc.ra118.006004" @default.
• W2913600982 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6463704" @default.
• W2913600982 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30770472" @default.
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