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• W3018159514 abstract "Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor–regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins. Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor–regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins. Chikungunya fever (CHIKF) is a re-emerging zoonotic disease caused by Chikungunya virus (CHIKV), a member of the genus Alphavirus, family Togaviridae (1Chen R. Mukhopadhyay S. Merits A. Bolling B. Nasar F. Coffey L.L. Powers A. Weaver S.C. Ictv Report ConsortiumICTV Virus Taxonomy Profile: Togaviridae.J. Gen. Virol. 2018; 99 (29745869): 761-76210.1099/jgv.0.001072Crossref PubMed Scopus (49) Google Scholar). The major symptoms of CHIKF are an acute febrile illness accompanied by arthralgia and rash (2Pialoux G. Gaüzère B.A. Jauréguiberry S. Strobel M. Chikungunya, an epidemic arbovirosis.Lancet Infect. Dis. 2007; 7 (17448935): 319-32710.1016/S1473-3099(07)70107-XAbstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar, 3Couderc T. Lecuit M. Chikungunya virus pathogenesis: from bedside to bench.Antiviral Res. 2015; 121 (26159730): 120-13110.1016/j.antiviral.2015.07.002Crossref PubMed Scopus (56) Google Scholar, 4Borgherini G. Poubeau P. Staikowsky F. Lory M. Le Moullec N. Becquart J.P. Wengling C. Michault A. Paganin F. Outbreak of chikungunya on Reunion Island: early clinical and laboratory features in 157 adult patients.Clin. Infect. Dis. 2007; 44 (17479933): 1401-140710.1086/517537Crossref PubMed Scopus (352) Google Scholar). Arthralgias may persist, and relapse, for weeks to months and even years, consequently reducing the quality of life of CHIKF patients (5Borgherini G. Poubeau P. Jossaume A. Gouix A. Cotte L. Michault A. Arvin-Berod C. Paganin F. Persistent arthralgia associated with chikungunya virus: a study of 88 adult patients on reunion island.Clin. Infect. Dis. 2008; 47 (18611153): 469-47510.1086/590003Crossref PubMed Scopus (293) Google Scholar, 6Schilte C. Staikowsky F. Staikovsky F. Couderc T. Madec Y. Carpentier F. Kassab S. Albert M.L. Lecuit M. Michault A. Chikungunya virus-associated long-term arthralgia: a 36-month prospective longitudinal study.PLoS Negl. Trop. Dis. 2013; 7 (23556021): e213710.1371/journal.pntd.0002137Crossref PubMed Scopus (267) Google Scholar). During the major outbreaks of CHIKF in the Indian Ocean islands in 2005, following the re-emergence of CHIKF in Kenya (7Chretien J.P. Anyamba A. Bedno S.A. Breiman R.F. Sang R. Sergon K. Powers A.M. Onyango C.O. Small J. Tucker C.J. Linthicum K.J. Drought-associated chikungunya emergence along coastal East Africa.Am. J. Trop. Med. Hyg. 2007; 76 (17360859): 405-40710.4269/ajtmh.2007.76.405Crossref PubMed Scopus (160) Google Scholar), CHIKF became disseminated worldwide via thousands of infected travelers (8Weaver S.C. Forrester N.L. Chikungunya: evolutionary history and recent epidemic spread.Antiviral Res. 2015; 120 (25979669): 32-3910.1016/j.antiviral.2015.04.016Crossref PubMed Scopus (220) Google Scholar). To date, CHIKF cases have been identified in over 60 countries, including outbreaks in Europe and the Americas (9Rezza G. Nicoletti L. Angelini R. Romi R. Finarelli A.C. Panning M. Cordioli P. Fortuna C. Boros S. Magurano F. Silvi G. Angelini P. Dottori M. Ciufolini M.G. Majori G.C. et al.Infection with chikungunya virus in Italy: an outbreak in a temperate region.Lancet. 2007; 370 (18061059): 1840-184610.1016/S0140-6736(07)61779-6Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar, 10Grandadam M. Caro V. Plumet S. Thiberge J.M. Souarès Y. Failloux A.B. Tolou H.J. Budelot M. Cosserat D. Leparc-Goffart I. Desprès P. Chikungunya virus, southeastern France.Emerg. Infect. Dis. 2011; 17 (21529410): 910-91310.3201/eid1705.101873Crossref PubMed Scopus (343) Google Scholar, 11Leparc-Goffart I. Nougairede A. Cassadou S. Prat C. de Lamballerie X. Chikungunya in the Americas.Lancet. 2014; 383 (24506907): 51410.1016/S0140-6736(14)60185-9Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Effective control measures for CHIKF are desired; however, no commercial vaccines or effective antiviral treatments currently exist. To develop new treatment strategies for CHIKV infection, a better understanding of the molecular mechanisms underlying CHIKV infection is required. The genome of CHIKV is a positive sense, single-stranded RNA composed of two open reading frames, encoding both nonstructural and structural polyproteins. The nonstructural proteins (nsPs), consisting of nsP1–4, are required for transcription and replication of viral RNA (12Solignat M. Gay B. Higgs S. Briant L. Devaux C. Replication cycle of chikungunya: a re-emerging arbovirus.Virology. 2009; 393 (19732931): 183-19710.1016/j.virol.2009.07.024Crossref PubMed Scopus (211) Google Scholar, 13Rupp J.C. Sokoloski K.J. Gebhart N.N. Hardy R.W. Alphavirus RNA synthesis and non-structural protein functions.J. Gen. Virol. 2015; 96 (26219641): 2483-250010.1099/jgv.0.000249Crossref PubMed Scopus (117) Google Scholar). The structural proteins (sPs), comprising the capsid (C) and envelope glycoproteins (E2 and E1), are the main constituents of virus particles and facilitate receptor binding and cellular entry (12Solignat M. Gay B. Higgs S. Briant L. Devaux C. Replication cycle of chikungunya: a re-emerging arbovirus.Virology. 2009; 393 (19732931): 183-19710.1016/j.virol.2009.07.024Crossref PubMed Scopus (211) Google Scholar, 14Voss J.E. Vaney M.C. Duquerroy S. Vonrhein C. Girard-Blanc C. Crublet E. Thompson A. Bricogne G. Rey F.A. Glycoprotein organization of chikungunya virus particles revealed by X-ray crystallography.Nature. 2010; 468 (21124458): 709-71210.1038/nature09555Crossref PubMed Scopus (398) Google Scholar). The CHIKV replication cycle begins with cellular attachment and entry via endocytosis (15van Duijl-Richter M.K. Hoornweg T.E. Rodenhuis-Zybert I.A. Smit J.M. Early events in chikungunya virus infection—from virus cell binding to membrane fusion.Viruses. 2015; 7 (26198242): 3647-367410.3390/v7072792Crossref PubMed Scopus (70) Google Scholar). The post-entry steps of CHIKV have not been fully characterized, but genome replication may take place both at the plasma membrane and type I cytopathic vacuoles (CPV-I), based on data obtained with other alphaviruses (e.g. Semliki Forest virus (SFV)) (16Spuul P. Balistreri G. Kääriäinen L. Ahola T. Phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of Semliki Forest virus replication complexes from the plasma membrane to modified lysosomes.J. Virol. 2010; 84 (20484502): 7543-755710.1128/JVI.00477-10Crossref PubMed Scopus (125) Google Scholar, 17Froshauer S. Kartenbeck J. Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.J. Cell Biol. 1988; 107 (2904446): 2075-208610.1083/jcb.107.6.2075Crossref PubMed Scopus (299) Google Scholar). Colocalization of SFV nsPs and newly synthesized viral RNAs is initially observed in spherule structures near the plasma membrane (16Spuul P. Balistreri G. Kääriäinen L. Ahola T. Phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of Semliki Forest virus replication complexes from the plasma membrane to modified lysosomes.J. Virol. 2010; 84 (20484502): 7543-755710.1128/JVI.00477-10Crossref PubMed Scopus (125) Google Scholar). Then SFV replication complexes are thought to be transported to CPV-I originating from late endosomes and lysosomes (16Spuul P. Balistreri G. Kääriäinen L. Ahola T. Phosphatidylinositol 3-kinase-, actin-, and microtubule-dependent transport of Semliki Forest virus replication complexes from the plasma membrane to modified lysosomes.J. Virol. 2010; 84 (20484502): 7543-755710.1128/JVI.00477-10Crossref PubMed Scopus (125) Google Scholar, 17Froshauer S. Kartenbeck J. Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes.J. Cell Biol. 1988; 107 (2904446): 2075-208610.1083/jcb.107.6.2075Crossref PubMed Scopus (299) Google Scholar). At the late stages of the viral infection, the newly synthesized E1/E2 glycoproteins are transported to the endoplasmic reticulum, trans-Golgi network (TGN), TGN-derived CPV-II, and finally the plasma membrane (18de Curtis I. Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells.Proc. Natl. Acad. Sci. U.S.A. 1988; 85 (3186706): 8052-805610.1073/pnas.85.21.8052Crossref PubMed Scopus (117) Google Scholar, 19Griffiths G. Quinn P. Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus.J. Cell Biol. 1983; 96 (6682112): 835-85010.1083/jcb.96.3.835Crossref PubMed Scopus (272) Google Scholar, 20Griffin D.E. Alphaviruses.in: Knipe D.M. Howley P.M. Fields Virology. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia2013: 651-686Google Scholar). Following the formation of nucleocapsid by binding of the alphaviral C protein and RNA (21Geigenmüller-Gnirke U. Nitschko H. Schlesinger S. Deletion analysis of the capsid protein of Sindbis virus: identification of the RNA binding region.J. Virol. 1993; 67 (8437233): 1620-162610.1128/JVI.67.3.1620-1626.1993Crossref PubMed Google Scholar, 22Perera R. Owen K.E. Tellinghuisen T.L. Gorbalenya A.E. Kuhn R.J. Alphavirus nucleocapsid protein contains a putative coiled coil α-helix important for core assembly.J. Virol. 2001; 75 (11119567): 1-1010.1128/JVI.75.1.1-10.2001Crossref PubMed Scopus (71) Google Scholar), virus particles are assembled and released from the plasma membrane (20Griffin D.E. Alphaviruses.in: Knipe D.M. Howley P.M. Fields Virology. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia2013: 651-686Google Scholar). Both CPV-I and -II have been observed in CHIKV-infected cells, and CHIKV has been shown to bud from the plasma membrane (12Solignat M. Gay B. Higgs S. Briant L. Devaux C. Replication cycle of chikungunya: a re-emerging arbovirus.Virology. 2009; 393 (19732931): 183-19710.1016/j.virol.2009.07.024Crossref PubMed Scopus (211) Google Scholar, 23Chen K.C. Kam Y.W. Lin R.T. Ng M.M. Ng L.F. Chu J.J. Comparative analysis of the genome sequences and replication profiles of chikungunya virus isolates within the East, Central and South African (ECSA) lineage.Virol. J. 2013; 10 (23721429): 16910.1186/1743-422X-10-169Crossref PubMed Scopus (28) Google Scholar). Although alphaviruses seem to be transported by a dynamic and unique subcellular trafficking machinery, little is known about the host factors involved in the intracellular CHIKV-trafficking processes (24Bernard E. Solignat M. Gay B. Chazal N. Higgs S. Devaux C. Briant L. Endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments.PLoS ONE. 2010; 5 (20628602): e1147910.1371/journal.pone.0011479Crossref PubMed Scopus (113) Google Scholar, 25Thomas S. Rai J. John L. Schaefer S. Pützer B.M. Herchenröder O. Chikungunya virus capsid protein contains nuclear import and export signals.Virol. J. 2013; 10 (23984714): 26910.1186/1743-422X-10-269Crossref PubMed Scopus (27) Google Scholar). A variety of enveloped viruses can exploit the cellular endosomal sorting complexes required for transport (ESCRT) in their infection steps (26Votteler J. Sundquist W.I. Virus budding and the ESCRT pathway.Cell Host Microbe. 2013; 14 (24034610): 232-24110.1016/j.chom.2013.08.012Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). In the ESCRT pathway, which is highly conserved across Eukarya, ubiquitinated proteins are sorted into the multivesicular bodies by deforming the membrane inward. A series of four distinct complexes (ESCRT-0, -I, -II, and -III) and VPS4 proteins are sequentially recruited for membrane deformation (27Henne W.M. Buchkovich N.J. Emr S.D. The ESCRT pathway.Dev. Cell. 2011; 21 (21763610): 77-9110.1016/j.devcel.2011.05.015Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar). Because HIV-1 was initially reported to engage the ESCRT pathway to acquire its envelope and pinch off viral particles during the budding step, other enveloped viruses have been shown to employ this pathway for their budding steps (26Votteler J. Sundquist W.I. Virus budding and the ESCRT pathway.Cell Host Microbe. 2013; 14 (24034610): 232-24110.1016/j.chom.2013.08.012Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Whereas VPS4 is required for the budding of a variety of viruses, SFV was shown to bud from cells independent of both VPS4 and ubiquitin, suggesting a possible ESCRT-independent budding process (28Taylor G.M. Hanson P.I. Kielian M. Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding.J. Virol. 2007; 81 (17913808): 13631-1363910.1128/JVI.01688-07Crossref PubMed Scopus (65) Google Scholar). However, the putative roles of ESCRT factors in infection of other alphaviruses have not been examined. The molecular mechanisms of viral trafficking by ESCRT factors remain to be elucidated. Herpes simplex virus, hepatitis C virus, dengue virus (DENV), and Japanese encephalitis virus utilize ESCRT factors, whereas their late assembly domains (L domains) known to serve as a viral motif to recruit ESCRT factors are still undiscovered (29Barouch-Bentov R. Neveu G. Xiao F. Beer M. Bekerman E. Schor S. Campbell J. Boonyaratanakornkit J. Lindenbach B. Lu A. Jacob Y. Einav S. Hepatitis C virus proteins interact with the endosomal sorting complex required for transport (ESCRT) machinery via ubiquitination to facilitate viral envelopment.MBio. 2016; 7 (27803188): e01456-1610.1128/mBio.01456-16Crossref PubMed Scopus (33) Google Scholar, 30Kumar S. Barouch-Bentov R. Xiao F. Schor S. Pu S. Biquand E. Lu A. Lindenbach B.D. Jacob Y. Demeret C. Einav S. MARCH8 ubiquitinates the hepatitis C virus nonstructural 2 protein and mediates viral envelopment.Cell Rep. 2019; 26 (30759391): 1800-1814.e510.1016/j.celrep.2019.01.075Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 31Pattanakitsakul S.N. Poungsawai J. Kanlaya R. Sinchaikul S. Chen S.T. Thongboonkerd V. Association of Alix with late endosomal lysobisphosphatidic acid is important for dengue virus infection in human endothelial cells.J. Proteome Res. 2010; 9 (20669987): 4640-464810.1021/pr100357fCrossref PubMed Scopus (26) Google Scholar, 32Chiou C.T. Hu C.A. Chen P.H. Liao C.L. Lin Y.L. Wang J.J. Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein.J. Gen. Virol. 2003; 84 (13679614): 2795-280510.1099/vir.0.19201-0Crossref PubMed Scopus (44) Google Scholar, 33Tabata K. Arimoto M. Arakawa M. Nara A. Saito K. Omori H. Arai A. Ishikawa T. Konishi E. Suzuki R. Matsuura Y. Morita E. Unique requirement for ESCRT factors in flavivirus particle formation on the endoplasmic reticulum.Cell Rep. 2016; 16 (27545892): 2339-234710.1016/j.celrep.2016.07.068Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 34Crump C.M. Yates C. Minson T. Herpes simplex virus type 1 cytoplasmic envelopment requires functional Vps4.J. Virol. 2007; 81 (17507493): 7380-738710.1128/JVI.00222-07Crossref PubMed Scopus (109) Google Scholar, 35Calistri A. Sette P. Salata C. Cancellotti E. Forghieri C. Comin A. Göttlinger H. Campadelli-Fiume G. Palù G. Parolin C. Intracellular trafficking and maturation of herpes simplex virus type 1 gB and virus egress require functional biogenesis of multivesicular bodies.J. Virol. 2007; 81 (17686835): 11468-1147810.1128/JVI.01364-07Crossref PubMed Scopus (85) Google Scholar, 36Pawliczek T. Crump C.M. Herpes simplex virus type 1 production requires a functional ESCRT-III complex but is independent of TSG101 and ALIX expression.J. Virol. 2009; 83 (19692479): 11254-1126410.1128/JVI.00574-09Crossref PubMed Scopus (106) Google Scholar). VPS4-independent ESCRT pathways have been described in DENV and Japanese encephalitis virus infection (33Tabata K. Arimoto M. Arakawa M. Nara A. Saito K. Omori H. Arai A. Ishikawa T. Konishi E. Suzuki R. Matsuura Y. Morita E. Unique requirement for ESCRT factors in flavivirus particle formation on the endoplasmic reticulum.Cell Rep. 2016; 16 (27545892): 2339-234710.1016/j.celrep.2016.07.068Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Among viruses, even within the same families, interactions between viruses and ESCRT factors appear different (26Votteler J. Sundquist W.I. Virus budding and the ESCRT pathway.Cell Host Microbe. 2013; 14 (24034610): 232-24110.1016/j.chom.2013.08.012Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). These studies suggest that the role of ESCRT factors in viral infection differs from virus to virus. Genome-wide screens using CHIKV-infected cells have identified a variety of host factors required for CHIKV infection; however, host trafficking factors have not been fully characterized (37Zhang R. Kim A.S. Fox J.M. Nair S. Basore K. Klimstra W.B. Rimkunas R. Fong R.H. Lin H. Poddar S. Crowe Jr., J.E. Doranz B.J. Fremont D.H. Diamond M.S. Mxra8 is a receptor for multiple arthritogenic alphaviruses.Nature. 2018; 557 (29769725): 570-57410.1038/s41586-018-0121-3Crossref PubMed Scopus (111) Google Scholar, 38Karlas A. Berre S. Couderc T. Varjak M. Braun P. Meyer M. Gangneux N. Karo-Astover L. Weege F. Raftery M. Schönrich G. Klemm U. Wurzlbauer A. Bracher F. Merits A. Meyer T.F. Lecuit M. A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.Nat. Commun. 2016; 7 (27177310): 1132010.1038/ncomms11320Crossref PubMed Scopus (55) Google Scholar). In this study, we have identified components of the ESCRT machinery required for CHIKV infection through siRNA screens. Subsequently, we have characterized the interaction of the identified ESCRT factors with CHIKV and a role for ESCRT factors in CHIKV infection. Our findings clearly demonstrated the importance of the host ESCRT trafficking machinery in the CHIKV replication cycle. To identify the trafficking genes required for CHIKV infection, we performed imaging-based siRNA screens. siRNAs against 73 trafficking genes belonging to the RAB, RHO, ARF/SAR, GOLGIN, and ESCRT families were selected for the screen. HEK293T cells were reverse-transfected with siRNA, cultured for 48 h, and then infected with CHIKV. The CHIKV infection rate was determined by dividing the number of CHIKV-positive cells by total cells. Three different siRNAs targeting the same gene product were examined independently. The heatmap was obtained by the relative cell count and relative infection rate in the presence of each siRNA (Fig. 1 and Table S1). For the siRNA screen, siRNA treatment was considered as a positive effect if the relative CHIKV infection rate of each siRNA treatment was <60% of control siRNA treatments and the relative cell count numbers of each siRNA treatment were >80% those of control siRNA treatments. Then we selected the gene as a hit if at least two of the three siRNAs targeting the same gene product had positive effects, to exclude potential off-target effects of each siRNA. As a result of the siRNA screen, we found 18 hits, including RAB (2 of 23), ARF/SAR (3 of 13), and ESCRT (13 of 29) genes, highlighted in red (Fig. 1). ARF3 and ARF4 in the ARF/SAR family were validated as hits, consistent with a previous report demonstrating the requirement of ARFs in CHIKV replication (39Zhang N. Zhang L. Key components of COPI and COPII machineries are required for chikungunya virus replication.Biochem. Biophys. Res. Commun. 2017; 493 (28962860): 1190-119610.1016/j.bbrc.2017.09.142Crossref PubMed Scopus (7) Google Scholar). Strikingly, 13 of 29 of the investigated ESCRT genes were related to the CHIKV infection, and those genes belonged to multiple ESCRT complexes, including ESCRT-0, -I, -II, -III, and VPS4. Moreover, siRNAs against some ESCRT genes, such as VPS37D and VPS4B, had great effects on CHIKV infection without the reduction of cell numbers. These results suggested that a series of ESCRT factors are involved in CHIKV infection. Because budding of SFV was considered independent of the ESCRT pathway, interactions between ESCRT factors and CHIKV infection have not been examined previously (28Taylor G.M. Hanson P.I. Kielian M. Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding.J. Virol. 2007; 81 (17913808): 13631-1363910.1128/JVI.01688-07Crossref PubMed Scopus (65) Google Scholar). Therefore, we focused our investigations on the ESCRT factors and examined their specific roles in CHIKV infection. To identify the ESCRT partners physically interacting with CHIKV proteins, we exogenously introduced HA-tagged host factors and FLAG-tagged CHIKV proteins and thereafter performed anti-HA co-immunoprecipitation (co-IP). Following the co-IP experiments with anti-HA antibody, precipitated CHIKV proteins combined with HA-tagged ESCRT proteins were detected with anti-FLAG antibody. We selected eight ESCRT-related factors in these studies. HGS, also known as Hrs, and STAM1 belong to the ESCRT-0 complex and act as a sorting machinery that recognizes ubiquitinated receptors (27Henne W.M. Buchkovich N.J. Emr S.D. The ESCRT pathway.Dev. Cell. 2011; 21 (21763610): 77-9110.1016/j.devcel.2011.05.015Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar). TSG101 (ESCRT-I) and ESCRT-associated proteins (ALIX and NEDD4 (E3 ubiquitin protein ligase)) are reported to bind to viral L domains (26Votteler J. Sundquist W.I. Virus budding and the ESCRT pathway.Cell Host Microbe. 2013; 14 (24034610): 232-24110.1016/j.chom.2013.08.012Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). VPS37D (ESCRT-I), CHMP4B (ESCRT-III), and VPS4B (VPS4 proteins) were considered as potential interactors with CHIKV proteins, because siRNA treatments against them markedly reduced CHIKV infection rates with little reduction of the relative cell count in the siRNA screen (Fig. 1). In addition, we also examined the arrestin domain–containing protein ARRDC1, which can recruit ESCRT factors at the plasma membrane in an L domain–dependent viral budding process (40Rauch S. Martin-Serrano J. Multiple interactions between the ESCRT machinery and arrestin-related proteins: implications for PPXY-dependent budding.J. Virol. 2011; 85 (21191027): 3546-355610.1128/JVI.02045-10Crossref PubMed Scopus (79) Google Scholar). A total of seven CHIKV proteins were examined (C, E1, E2, and nsP1–4). Immunoblot analyses of IP fractions showed that HGS strongly interacted with E1, E2, and nsP4 (Fig. 2, A and B). The co-IP results of nsP3 fractions were excluded, because exogenously expressed FLAG-nsP3 was nonspecifically precipitated with anti-HA antibody (data not shown). The obtained results were confirmed by reciprocal immunoprecipitation with FLAG-tagged CHIKV proteins and detection of HA-tagged ESCRT proteins with anti-HA antibody. Co-IP results showed that HGS interacted with E1, E2, and nsP4 (Fig. 2, C and D). In addition, specific bands for NEDD4 were detected in the nsP1, nsP3, and nsP4 fractions, and TSG101 bands were detected in the nsP3 and nsP4 fractions (Fig. 2D). We excluded the results of VPS37D and CHMP4B fractions, because exogenously expressed HA-VPS37D and CHMP4B were nonspecifically precipitated with anti-FLAG antibody (data not shown). These results showed that HGS interacted with both sPs and nsPs of CHIKV, suggesting that HGS may have a critical role in multiple steps of CHIKV infection. Then we examined the interaction of HGS and CHIKV E2 in CHIKV-infected cells. HEK293T cells were transfected with either HA-HGS– or HA-STAM1–expressing plasmid and thereafter inoculated with CHIKV. HA-tagged HGS but not STAM1 was recovered from the immunoprecipitates of CHIKV-infected cells with anti-CHIKV E2 antibody (Fig. 3A). These results suggest that CHIKV E2 interacts with HGS in CHIKV-infected cells. To further characterize the interactions of HGS with CHIKV proteins, we searched the HGS region responsible for binding to CHIKV proteins using truncated HGS mutants lacking amino acid residues 1–277, 278–527, or 528–777 (Δ1–277, Δ278–527, or Δ528–777, respectively). HA-tagged truncated HGS mutants were immunoprecipitated, followed by detection of CHIKV proteins. The IP fractions indicated that HGS Δ278–527 had markedly reduced binding ability to CHIKV E1, E2, and nsP4 compared with full-length HGS protein (WT), Δ1–277, and Δ528–777 (Fig. 3B). The reciprocal experiments confirmed that HGS Δ278–527 also failed to bind to CHIKV E1, E2, and nsP4 (Fig. 3C). These results suggested that the amino acid residues 278–527 of HGS play an important role in binding to CHIKV sPs and nsPs. Next, we examined the interactions of HGS and CHIKV in CHIKV-infected cells by confocal microscopy. As commercially available antibodies failed to visualize endogenous HGS in Huh-7 cells, probably due to the low level of endogenous HGS protein that has been reported previously (41Chou S.F. Tsai M.L. Huang J.Y. Chang Y.S. Shih C. The dual role of an ESCRT-0 component HGS in HBV transcription and naked capsid secretion.PLoS Pathog. 2015; 11 (26431433): e100512310.1371/journal.ppat.1005123Crossref PubMed Scopus (38) Google Scholar), we generated Huh-7 cells stably expressing HGS (HGS-Huh-7 cells). We then confirmed that the distribution of HGS was similar to the distribution of endogenous HGS in HeLa cells, as shown in the previous report (42Komada M. Masaki R. Yamamoto A. Kitamura N. Hrs, a tyrosine kinase substrate with a conserved double zinc finger domain, is localized to the cytoplasmic surface of early endosomes.J. Biol. Chem. 1997; 272 (9252367): 20538-2054410.1074/jbc.272.33.20538Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar); the dotlike signals of HGS were detected in the cytoplasm of mock-infected HGS-Huh-7 cells (Fig. 4A). In addition, the morphology and growth activity of HGS-Huh-7 cells was similar to the parent HGS cells. Using HGS-Huh-7 cells, we first examined the localization of HGS and CHIKV E2 at 12 h post-infection (hpi) of CHIKV. As shown in the middle and bottom panels of Fig. 4A, colocalization of HGS and CHIKV E2 was observed in the cytoplasm of CHIKV-infected HGS-Huh-7 cells. Next, we examined colocalization of the HGS and CHIKV replication site. To achieve this, we applied anti-dsRNA antibody, as dsRNA is exclusively expressed in the CHIKV replication site in CHIKV-infected cells (43Remenyi R. Gao Y. Hughes R.E. Curd A. Zothner C. Peckham M. Merits A. Harris M. Persistent replication of a chikungunya virus replicon in human cells is associated with presence of stable cytoplasmic granules containing nonstructural protein 3.J. Virol. 2018; 92 (29875241): e00477-1810.1128/JVI.00477-18Crossref PubMed Scopus (16) Google Scholar). Colocalization of HGS and dsRNA in the cytoplasm of CHIKV-infected cells was confirmed (middle and bottom panels, Fig. 4B). Taken together, the ESCRT protein, HGS, colocalized with CHIKV structural protein and replication sites in CHIKV-infected cells. Thus, we next examined CH" @default.
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• W3018159514 date "2020-06-01" @default.
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• W3018159514 title "Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle" @default.
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